EMBO Conference on Protein Synthesis and Translational Control

Abstracts of papers presented at the 

<strong>EMBO</strong> <strong>Conference</strong> on Protein Synthesis 

and Translational Control 

EMBL Heidelberg, 9 - 13 September 2009 

Scientific Organisers: 

Anne Ephrussi, EMBL Heidelberg, Germany 

Matthias Hentze, EMBL Heidelberg, Germany 

Marina Rodnina, MPI Göttingen, Germany 

Nahum Sonenberg, McGill University, Canada 

<strong>Conference</strong> Organiser: 

Bettina Schäfer, EMBL Heidelberg, Germany 

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal 

communication and should be cited as such only with the consent of the author.

Content 

Agenda i 

Posters A-Z xv 

Speaker Abstracts 1 

Poster Abstracts 68 

Authors Index 285 

List of Participants 321 

Useful Telefonnumbers 362 

Map of EMBL Campus 363

15:00 - 18:00 Registration 

18:00 - 19:00 Dinner 

19:00 - 19:15 Welcome 

19:15 - 20:00 Judith Kimble 

20:00 - 20:45 David Bartel 

DAY 1, 9 SEPTEMBER 2009 

i 

Agenda 

University of Wisconsin-Madison, United States of 

America 

Translational control and stem cells: 

lessons from the C. elegans germline 1 

MIT/Whitehead Institute/HHMI, United States of 

America 

MicroRNAs 2 

20:45 - 21:15 Coffee Break 

21:15 - 22:00 Reinhard Luehrmann 

MPI fuer Biophysikalische Chemie, Germany 

At the heart of the spliceosome 3 

22:00 - 00:00 Get-together 

DAY 2, 10-SEPTEMBER 2009 

09:00 - 12:30 Session 1: Translation Initiation 

09:00 - 09:30 Jon Lorsch 

Johns Hopkins University School of Medicine, 

United States of America 

Reconstitution and Analysis of 

Cap-stimulated mRNA Recruitment to the 

43S Pre-initiation Complex 4

<strong>EMBO</strong> <strong>Conference</strong> on Protein Synthesis and Translational Control 

EMBL Heidelberg, 9 – 13 September 2009 

09:30 - 09:45 Adesh Saini 

NICHD, NIH, United States of America 

Structural elements in eIF1A regulate AUG 

selection by controlling distinct modes of 

initiator binding to the preinitiation 

complex 5 

09:45 - 10:00 Martin Jennings 

10:00 - 10:15 Akira Fukao 

University of Manchester, United Kingdom 

eIF5 acts as a GDP dissociation inhibitor in 

addition to its GAP function 6 

Kobe University, Japan 

10:15 - 10:30 Franck Martin 

Enhancement of cap-dependent translation 

by the ELAV protein HuD: A novel function 

of HuD which is eIF4A- and 

poly(A)-dependent 7 

Université de Strasbourg, France 


11:00 - 11:15 Rivka Dikstein 

Tethering of ribosomes downstream of 

translational start codon drives translation 

of histone H4 8 

Weizmann Institute of Science, Israel 

Characterization of TISU, a translation 

initiator specific to mRNAs with extremely 

short 5’UTR 9 

11:15 - 11:30 Christine Luttermann 

Friedrich-Loeffler-Institut, Germany 

The importance of inter- and 

intramolecular base pairing for translation 

reinitiation on a eukaryotic bicistronic 

mRNA 10 

ii

11:30 - 11:45 Anna Maria Giuliodori 

11:45 - 12:00 Dieter Wolf 

12:00 - 12:15 Helena Firczuk 

Laboratory of Genetics, Department of Biology 

MCA, University of Camerino,, Italy 

iii 

Agenda 

Translational regulation of cold-shock 

gene expression 11 

Burnham Institute for Medical Research, United 

States of America 

The eIF3 interactome reveals the 

translasome, a supercomplex linking 

protein synthesis and degradation 

machineries 12 

Manchester Interdisciplinary Biocentre, The 


Comprehensive rate control analysis of the 

eukaryotic translation pathway 13 

12:15 - 12:30 Nicholas Ingolia 

12:30 - 14:00 Lunch 

University of California, San Francisco, United 


Genome-wide analysis of in vivo 

translation with single-nucleotide 

resolution 14 

14:00 - 16:30 Session 2: Translation elongation and 

termination 

14:00 - 14:30 Terri Kinzy 

UMDNJ Robert Wood Johnson Medical School, 


ADP-ribosylation of eukaryotic elongation 

factor 2 by bacterial toxins and its effects 

on translation elongation in vivo and in 

vitro 15



14:30 - 14:45 Alena Paleskava 

14:45 - 15:00 James Munro 

Max Planck Institute for Biophysical Chemistry, 

Germany 

Unusually tight binding of Sec-tRNASec to 

the elongation factor SelB due to the 

specific recognition of the selenocysteyl 

group by the GTP-bound form 16 

Weill Cornell Medical College, United States of 

America 

Single-molecule observations of 

rate-limiting conformational events during 

ribosomal translocation 17 

15:00 - 15:15 Shashi Bhushan 

15:15 - 15:30 C. Axel Innis 

15:30 - 15:45 Kristen Bartoli 

15:45 - 16:00 Erik Böttger 


Gene Cemter, LMU Munich, Germany 

Visualization of nascent chains in the 

ribosomal exit tunnel: Implication for 

secondary structure formation 18 

Yale University, United States of America 

Shedding Light Onto Nascent 

Chain-Mediated Translational Stalling 19 

University of Pittsburgh, School of Medicine, United 


Novel Role for Mitotic Microtubule Motor 

Protein Eg5 in Protein Translation 20 

Institute of Medical Microbiology, Switzerland 

Aminoglycoside Ototoxicity and Synthesis 

of New Compounds with Altered 

Drug-Target Interaction 21 

iv

16:30 - 18:30 Session 3: Non-coding RNAs in translation 

16:30 - 17:00 Witold Filipowicz 

Friedrich Miescher Institute for Biomedical 

Research, Germany 

v 

Agenda 

Mechanism of the HuR-mediated reversal 

of miRNA repression in human cells 22 

17:00 - 17:15 Elisa Izaurralde 

17:15 - 17:30 Thomas Preiss 

Max Planck Institute for Developmental Biology, 

Germany 

A C-terminal silencing domain in GW182 

family proteins is essential for miRNA 

function in animal cells 23 

VCCRI, Australia 

17:30 - 17:45 Incheol Ryu 

Identifying miRNA targets through 

changes in mRNA poly(A) tail length 24 

POSTECH, Republic of Korea 

Eukaryotic Translation Initiation Factor 4G 

Mediates MicroRNA-Regulated 

Translational Gene Silencing 25 

17:45 - 18:00 Ania Wilczynska 

Univerisity of Cambridge, United Kingdom 

Investigating the microRNA pathway in 

Xenopus oocytes 26 

18:00 - 18:15 Anne-Catherine Prats 

Inserm U858, France 

FGF1 induction in myogenesis depends on 

novel cross-talks between IRES, promoter 

and 3’UTR 27



18:15 - 18:30 William Merrick 

Case Western Reserve University, United States of 

America 

Possible mechanism of regulation of 

IRES-mediated expression by eIF2A 28 

18:30 - 20:00 Dinner 

20:00 - 22:00 Poster Session I 

22:00 - 00:00 Wine & Cheese 

DAY 3, 11-SEPTEMBER 2009 

09:00 - 12:30 Session 4: Translation in development and the 

CNS 

09:00 - 09:30 Christine Holt 

09:30 - 09:45 Raúl Méndez 

University of Cambridge, United Kingdom 

Sub-cellular profiling reveals distinct and 

dynamic repertoire of growth cone mRNAs 29 

Centre for Genomic Regulation (CRG), Spain 

CPEB1 regulates the expression of CPEB4 

to complete meiosis 30 

09:45 - 10:00 Howard Lipshitz 

University of Toronto, Canada 

10:00 - 10:15 Maria Barna 

Control of mRNA translation and stability 

during early Drosophila development 31 

University of California, San Francisco, United 


Translational specificity of ribosomal 

proteins regulates vertebrate embryonic 

development 32 

vi

10:15 - 10:30 Michael Sheets 

Univ. of Wisconsin Dept. of Biomol. Chem., United 


vii 

Agenda 

Spatially regulated translation of the xCR1 

mRNA in xenopus embryos by poly (A) 

independent mechanism 33 


11:00 - 11:15 Sebastian Baumann 

Max Planck Institute for Terrestrial Microbiology, 

Germany 

Microtubule-dependent mRNA transport 

during pathogenic development in Ustilago 

maydis 34 

11:15 - 11:30 Peter Lukavsky 

MRC LMB, United Kingdom 

A’-form RNA helices drive 

microtubule-based mRNA transport in 

Drosophila 35 

11:30 - 11:45 Greco Hernandez 

McGill University, Canada 

11:45 - 12:00 Ana Villlalba 

Mextli is a novel eIF4E-binding protein 

from Drosophila 36 

FUNDACIO PRIVADA CENTRE DE REGULACIO 

GENOMICA, Spain 

A novel, non-canonical mechanism of 

cytoplasmic polyadenylation in Drosophila 37 

12:00 - 12:15 Kobi Rosenblum 

Haifa University, Israel 

Translational Control in the Gustatory 

Cortex Determines the Stability of a Taste 

Memory 38



12:15 - 12:30 Kent Duncan 

12:30 - 14:00 Lunch 

EMBL, Germany 

The SXL-UNR Corepressor Complex Uses 

a Novel PABP-Mediated Mechanism to 

Inhibit Ribosome Recruitment to msl-2 

mRNA 39 

14:00 - 17:30 Session 5: Translation factors & complexes, and 

their function in health and disease 

14:00 - 14:30 Fatima Gebauer 

Fundacio Privada Centre de Regulaciao Genomica, 

Spain 

Regulatory networks controlled by 

Drosophila UNR 40 

14:30 - 14:45 Shelton Bradrick 

14:45 - 15:00 Michal Shapira 

15:00 - 15:15 Gregory Boel 

Duke University, United States of America 

Identification of gemin5 as a novel 

cap-binding protein that associates with 

coding and non-coding RNAs 41 

Ben Gurion University of the Negev, Israel 

Evolutionary diversity of the 

trypanosomatid cap4-binding complex – a 

potential drug target against 

trypanosomatids? 42 

Columbia University, United States of America 

YjjK, a member of the ATP binding 

cassette superfamily is a novel 

transcriptional factor 43 

viii

15:15 - 15:30 Lyubov Ryabova 

Institut de Biologie Moléculaire des Plantes (IBMP), 

UPR CNRS 2357, France 

ix 

Agenda 

Function of RISP in virus-induced 

translation reinitiation 44 

15:30 - 15:45 Philip Farabaugh 

University of Maryland Baltimore County, United 


Effects of haploinsufficiency of ribosomal 

protein and assembly factor genes on 

cellular physiology mediated by RACK1 45 


16:15 - 16:30 Vitaly Polunovsky 

University of Minnesota, United States of America 

Translational Control of Malignancy in the 

Murine Models Epithelial Carcinogenesis: 

The Role of the Translational Repressors 

4E-BPs 46 

16:30 - 16:45 Robert Rhoads 

LSU Health Sciences Center, United States of 

America 

Effects on translation initiation and 

malignant transformation of MMTV 

insertion into the eIF3e gene 47 

16:45 - 17:00 Robert Schneider 

NYU School of Medicine, United States of America 

AUF1 links the chronic inflammatory 

response to telomere maintenance, 

premature aging and tumorigenesis 

through control of short-lived mRNA 

stability 48



17:00 - 17:15 Harald König 

Forschungszentrum Karlsruhe GmbH, Germany 

Interfering with translation of transcripts 

enhances their splicing 49 

17:15 - 17:30 Michael Kiebler 

Medical University of Vienna, Austria 

Functional Characterization of Neuronal 

RNA granules and their role in dendritic 

RNA localization 50 

17:30 - 20:00 Dinner 

20:00 - 22:00 Poster Session II 

22:00 - 00:00 Wine & Cheese 


09:00 - 11:15 Session 6: mRNA stability and 

nonsense-mediated decay 

09:00 - 09:30 Elena Conti 

Max Planck Institute of Biochemistry, Germany 

Structural studies of nonsense mediated 

mRNA decay 51 

09:30 - 09:45 Oliver Mühlemann 

09:45 - 10:00 Fulvia Bono 

University of Bern, Switzerland 

SMG6-mediated endonucleolytic cleavage 

of nonsense mRNA in human cells 52 

Max Planck of Dev. Biology, Germany 

Structural analysis of Mago-Y14 import 

receptor 53 

x

10:00 - 10:15 Gabriele Neu-Yilik 

MMPU EMBL/University of Heidelberg, Germany 

xi 

Agenda 

Premature termination codons within exon 

1 of the human beta-globin mRNA create 

short ORFs permissive for reinitiation 54 

10:15 - 10:30 Jonathan Dinman 

10:30 - 10:45 Sevim Ozgur 

10:45 - 11:00 Felix Tritschler 

University of Maryland, United States of America 

mRNA destabilization by programmed 

ribosomal frameshifting and its effects on 

telomere maintenance and aging in yeast 55 

German Cancer Research Center, Germany 

Human Pat1 Controls the Assembly of 

Processing-Bodies and Promotes mRNA 

Degradation 56 

Max-Planck-Institute for Developmental Biology, 

Germany 

A metazoan-specific DCP1 C-terminal 

extension is required for the assembly of 

active mRNA decapping complexes in 

human cells 57 

11:00 - 11:15 Markus Landthaler 

BIMSB, Germany 

PURE-CLIP - Transcriptome-wide 

identification of RNA targets and binding 

sites of RNA-binding proteins 58 

11:15 - 13:00 Poster Session III 

13:00 - 18:30 Lunch & Free Time 

18:30 - 19:30 Drinks 

19:30 - 01:00 Banquett Dinner & Party




09:15 - 12:15 Session 7: Structure and function of the 

ribosome 

09:15 - 09:45 Jamie Cate 

09:45 - 10:00 Stefano Marzi 

UC Berkeley, United States of America 

Insights into protein synthesis from 

structures of the E. coli ribosome 59 

IBMC Strasbourg, France 

A dynamic view of translation initiation and 

its control by structured mRNAs in 

bacteria 60 

10:00 - 10:15 Claudio Gualerzi 

University of Camerino, Italy 

10:15 - 10:30 Joachim Frank 


11:00 - 11:15 Lasse Jenner 

Mechanism of ribosomal recruitment of 

fMet-tRNA by bacterial translation initiation 

factor IF2 61 

HHMI, Columbia University, United States of 

America 

Structural Discrimination Between Cognate 

and Near-cognate Ternary Complexes By 

the Ribosome 62 

CERBM-GIE / IGBMC, France 

Crystal structure of the ribosome 

containing three tRNAs 63 

xii

11:15 - 11:30 Rebecca Voorhees 

11:30 - 11:45 Niels Fischer 

Medical Research Council: Laboratory of Molecular 

Biology, United Kingdom 

xiii 

Agenda 

Insights into substrate stabilization from 

structural studies of the peptidyl 

transferase center of the intact 70S 

ribosome 64 

Max Planck Institute for Biophysical Chemistry, 

Germany 

The trajectory of tRNA movement through 

the ribosome visualized by time-resolved 

electron cryomicroscopy 65 

11:45 - 12:00 Christian Spahn 

Charite - Universitätsmedizin Berlin, Germany 

Visualization of conformational modes of 

ribosomal complexes by multi-particle cryo-EM 66 

12:00 - 12:15 Rebecca Kohler 

ETH Zuerich, Switzerland 

YidC and Oxa1 Form Dimeric Insertion 

Pores on the Translating Ribosome 67 

12:15 - 12:30 Lunch & Departure

xv 

Posters A-Z 

Achsel, Tilmann 

Identification and characterisation of the human Pat1: a novel deadenylation factor 68 

Akbergenov, Rashid Z. 

rRNA Sequence Polymorphism within the Bacterial Domain and Susceptibility to Drugs 

Targeting Protein Synthesis 69 

Anderson, Ross C. 

Investigating the Expression and Functions of the Poly(A)-Binding Protein Family Within 

Mammalian Gonads 70 

Andreev, Dmitry E. 

Translation machinery can efficiently scan through the highly structured 

5’ UTR of Apaf-1 mRNA containing putative IRES 71 

Arribere, Joshua A. 

A comprehensive analysis of environmentally regulated yeast 

5'UTR variants: annotation and insights into functionality in translation regulation 72 

Badura, Michelle 

DNA damage mediates a novel control of translation by signalling through the DNA 

damage response complex to control mTOR activity and 4E-BP1 stability 73 

Balagopal, Vidya 

Stm1 modulates translation and mRNA decay in Saccharomyces cerevisiae 74 

Bastide, Amandine 

Investigation of Translational Regulation during Cold-Shock 75 

Belsham, Graham J. 

Themes and variations in HCV-like IRES elements 76 

Benyumov, Alexey 

Translational regulation of the epithelial -TO-mesenchymal transition: insights from 

mesoderm restriction in zebrafish gastrulation 77 

Bhattacharya, Rumpa 

Regulation of mRNA transaltion during recovery from heat shock 78 

Boehringer, Daniel 

Co-translational folding and membrane insertion of newly synthesized polypeptides 79 

Bottley, Andrew 

Translational profiling reveals an important role for eIF4A in the regulation of specific 

mRNAs 80 

Brogna, Saverio 

Visualization of ribosome subunits interaction in cells 81 

Burgess, Hannah M. 

PABP1 and PABP4 relocalise to the nucleus following UV irradiation 82



Bushell, Martin D. 

Localisation of MicroRNA-repressed MRNAS to P-Bodies prevents an initiation 

independent viral IRES from overcomingMicroRNA repression 83 

Byström, Anders S. 

Wobble uridine modifications in yeast 84 

Cajigas, Iván J. 

Regulation of mRNA stability in the hippocampus 85 

Calkhoven, Cornelis F. 

Discovery of Translationally Active Small Molecules that Inhibit Proliferation of Cancer 

Cells Using Translation Re-initiation Index (TRI) Determination 86 

Casanova, Claudia M. 

High-throughput siRNA screen to identify regulators of VEGF IRES translation 87 

Cencic, Regina 

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol 88 

Chavatte, Laurent 

Structural elements from the SECIS that determine UGA/selenocysteine recoding 

efficiency 89 

Chen, Changchun 

Defects in tRNA Modification Associate with Neurological and Developmental 

Dysfunctions in Caenorhabditis elegans Elongator Mutants 90 

CHIEN, Wei Wen 

p16INK4a inhibits CDK1 expression in MCF7 cells via the microRNA pathway 91 

Chirkova, Anna 

How does 23S rRNA contribute to tRNA movement through the ribosome? 92 

Chung, Betty Y. 

A small slip back for the ribosome reveals more than just moving forward 93 

Clarkson, Bryan K. 

Examining Functions of eIF4G Isoforms in Saccharomyces cerevisiae 94 

Cobbold, Laura C. 

A mutant form of the c-myc IRES has increased interactions with the ITAFs PTB and 

YB-1 95 

Coordes, Britta 

Translational regulation by phosphorylation of ribosomal proteins 96 

Cuchalova, Lucie 

Yeast eIF3g promotes resumption of scanning of post-termination ribosomes as a part of 

the GCN4 reinitiation mechanism whereas eIF3i stimulates processivity of scanning 97 

xvi

xvii 

Posters A-Z 

Curran, Joseph 

Impact of alternative 5'UTRs on the translational expression of the human 

mdm2 (hdm2) and elk-1 mRNAs 98 

Czech, Andreas 

Influence of stress conditions on tRNA composition and translation 

efficiency 99 

Darzynkiewicz, Edward B. 

Enzymatically Stable and Translationally Highly Effective 

Phoshorothioate Cap Analogs 100 

Daugeron, Marie-Claire 

The yeast GTPases Rbg1 and Rbg2 are implicated in translation 101 

Dave, Richa 

Direct observations of the tetrameric ribosomal stalk protein L12 in the 

multistep process of tRNA selection 102 

David, Alexandre 

Aminoacyl synthetases Reveal Compartmentalization of Protein Translation 103 

De Colibus, Luigi 

Structural studies of Nhm1, a key enzyme in the nuclear and cytoplasmic 

metabolism of RNA 104 

de Melo Neto, Osvaldo P. 

Two eIF4G Homologues from Trypanosomatids Display Functional 

Properties Compatible With Roles In Two Diverged eIF4F Complexes 105 

de Moor, Cornelia H. 

The polyadenylation inhibitor cordycepin disrupts mTOR signalling 106 

De Rubeis, Silvia 

CYFIP1, a neuronal eIF4E-BP, links local translational regulation to spine remodeling: 

insights into the Fragile X Syndrome 107 

de Vries, Sebastian 

The role of 3’UTR binding factors in VEGF IRES mediated mRNA translation 108 

Deborah, Silvera 

Translational regulation of the epithelial to mesenchymal transition in 

inflammatory breast cancer 109 

Desterro, Joana P. 

Auto-regulation of the heterodimeric splicing factor U2AF 110 

Dever, Thomas E. 

Requirement for Kinase-induced Conformational Change in eIF2a Restricts 

Phosphorylation of Ser51 111



Dobrikova, Elena 

Association of herpes simplex virus (HSV-1) proteins ICP27 and UL47 with 

polyA-binding protein (PABP) 112 

Dobson, Tara 

Identifying Mechanisms Contributing to (Over)Expression of 

Aurora A Kinase 113 

Doller, Anke 

PKC delta coordinates RNA-binding and export of nuclear HuR via a dual 

phosphorylation on serine 221 and 318 114 

Doronina, Victoria A. 

Dissecting ‘stop – carry on’ translating recoding 115 

Dougherty, Jonathan D. 

Disruption of processing body (PB) formation by a plus-strand RNA virus 116 

Dunkle, Jack A. 

Structures of the ribosome in an intermediate state of translocation 117 

Duss, Olivier 

NMR Study of the 60kDa Complex between the ncRNA RsmZ and the Bacterial Global 

Regulatory Protein RsmE 118 

Eldad, Naama 

RNA Polymerase II subunits link transcription and mRNA decay 

to translation 119 

Eliseeva, Irina 

On the mechanism of YB-1 mRNA translation inhibition by polyadenylation 120 

Endo, Kei 

Post-transcriptional gene repression induced by an artificial cis 

element of RNA aptamer to mammalian initiation factor eIF4AIII 121 

Fedyunin, Ivan 

Impact of altered translation by modified tRNA-profile on the co-translational folding 122 

Filbin, Megan E. 

Importance of Long-Range Communication Between Two Domains 

in the HCV IRES for Formation and Fidelity of 80S Ribosomes 123 

Fischer, Jeffrey 

Mechanistic insight into the function of the universally conserved GTPASE HFLX from 

ESCHERICHIA COLI 124 

Flanagan, John 

Observation of distinct A/P hybrid-state tRNAs in translocating ribosomes 125 

xviii

xix 

Posters A-Z 

Fox, Paul L. 

Cdk5- and p70 S6K-mediated, 2-site phosphorylation of a tRNA 

synthetase induces transcript-selective inhibition of translation 126 

Frugier, Magali 

Particularities of Plasmodial translational machinery: Structural and functional 

analysis of specific insertions in Plasmodium falciparum Aspartyl-tRNA synthetase 127 

Gamberi, Chiara 

Bicaudal-C and Ccr4 repress nanos expression during oogenesis 128 

Gehring, Niels H. 

Disassembly of exon junction complexes by the ribosome-associated 

protein PYM 129 

Gilbert, Robert J. 

Eukaryotic ribosome structure in the light of cellular differentiation 130 

Goetz, Christian 

eIF4E Status Controls cap-Independent Translation and Oncolysis of a Poliovirus 

Recombinant 131 

Golovina, Anna 

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically 

modifies A37 of tRNA1Val (cmo5UAC) 132 

Gorgoni, Barbara 

Multiple Poly(A)-Binding Proteins are essential for 

Xenopus laevis development 133 

Grosso, Stefano 

mTORc1 inhibition does not repress initiation of translation in cancer cell models 134 

Guarneros, Gabriel 

Translation termination factors (RF1, RF2, and RF3), ribosome recycling factor (RRF) 

and elongation factor (EF-G) rescue ribosomes stalled at sense codons 135 

Guo, Huili 

Investigating the effect of microRNAs on ribosome occupancy 136 

Haas, Gabrielle 

Analysis of the interactions and P-body localization of decapping activators in metazoan137 

Hauryliuk, Vasili V. 

Interaction framework among eRF1 / eRF3 / PABP and G nucleotides: complete 

thermodynamical analysis 138 

Heurgué-Hamard, Valérie 

Function of eRF1 methyltransferase subunits Mtq2p and Trm112p 

in ribosome biogenesis in S. cerevisiae 139



Hirnet, Juliane 

Cap-independent translation of poliovirus is affected by a 

neuron-specific microRNA 140 

Howe, Philip H. 

Transforming Growth Factor-ß (TGFß)-mediated Transcript Selective Translational 

Activation of EMT Inducer mRNAs 141 

Igreja, Cátia 

Multiple roles for CUP in translational repression 142 

Inada, Toshifumi 

40S ribosome-bound RACK1 functions in nascent peptide-dependent 

Translation arrest 143 

Jang, JC 

The Arabidopsis tandem zinc finger protein AtTZF1 traffics between the nucleus 

and cytoplasmic foci and affects development and hormone response 144 

Jaroszynski, Lukasz 

Translational control of DDX3Y gene transcripts by miRNA binding 

in 3’ UTR 145 

Jemielity, Jacek 

Tetraphosphate mRNA Cap Analogues with high affinity for eIF4E and increased stability 

toward decapping enzymes 146 

Jinek, Martin 

Biochemical and structural studies of the microRNA-mediated translational repression 147 

Jopling, Catherine L. 

Regulation of translation by microRNA-122 binding to the hepatitis C virus 5' untranslated 

region 148 

Jovanovic, Marko 

A Quantitative Targeted Proteomics Approach to Identify microRNA 

Targets in C. elegans 149 

Kafasla, Panagiota 

Mapping the orientation of PTB binding to picornavirus IRESs 150 

Karim, Zhala 

The EF4 (LepA) Effect on Reverse Translocation 151 

Karimian Pour, Navaz 

Translational Control of ApoB mRNA: Insulin Modulation via Localization in 

Cytoplasmic P Bodies 152 

Kelleher, Ray 

The functional roles of microRNAs in the developing and adult brain 153 

xx

xxi 

Posters A-Z 

Khoshnevis, Sohail 

biophysical studies of interactions of eIF3i within the eIF3 complex 154 

Knutsen, Jon Halvor J. 

A possible link between cell growth and the cell cycle: eIF2 alpha phosphorylation 155 

Komarova, Anastassia V. 

Measles virus ribonucleoprotein core and cellular translational machinery 156 

Kowalska, Joanna 

Boranophosphate Analogs of mRNA 5' end 157 

Kramer, Günter 

The function of the chaperone trigger factor in co-translational folding of proteins 158 

Krebber, Heike 

The yeast mRNA export factor Npl3p functions in translation initiation 159 

Krjuchkova, Polina N. 

Stop codon recognition sites in eRF1 160 

Kropiwnicka, Anna 

Binding affinities of eIF4E and eIF(iso)4E from Arabidopsis thaliana for mRNA cap 

analogues 161 

Kubacka, Dorota 

Investigating the cap-binding ability of the ovary-specific Xenopus eIF4E1b 162 

Lammich, Sven 

The Expression of the alpha-secretase ADAM10 is regulated via its 5'UTR 163 

Lawrence, Marlon 

The Extended Loops of Ribosomal Proteins L4 and L22 are Not Essential for 

Ribosome Function, Cell Survival, or Peptide-Mediated Translational Arrest 164 

Leichter, Michael 

A potential role for the survival of motorneuron complex and methylosome in the 

assembly of selenoprotein mRNAs 165 

Lerner, Rachel S. 

SLIP1 Plays a Role in Transport, Translation, and Degradation of Histone mRNA 166 

Lescure, Alain 

Homozygous mutations in the SEPN1 gene affecting insertion of selenocysteine and 

causing rigid spine muscular dystrophy 167 

Leung, Kin-Mei 

Live imaging of ß-actin mRNA transport in retinal axons and growth cones 168



Leutz, Achim 

Physiological relevance of the C/EBPbeta uORF 169 

Liberman, Noa 

DAP5 - A Translation Initiation Factor that mediates Cap-Independent Translation 170 

Lin, Chien-Ling 

The Nuclear Experience of CPEB: Implications for RNA Processing and Translational 

Control 171 

Lin, Jing-Yi 

Mechanism of negative regulation by Far upstream element 

binding protein 2 interacts with enterovirus 71 internal ribosomal entry site 172 

Lin, Zhaoru 

Probing the Mysteries of Ribosomal Frameshifting using Antisense Oligonucleotides 173 

Liu, Weizhi 

Structural Insights into Parasite eIF4E Dual Binding Specificity 

for Monomethyl and Trimethylguanosine mRNA Caps 174 

Lopez-Lastra, Marcelo 

The 5’UTR of the MMTV mRNA exhibits cap-independent 

translation initiation 175 

Loreni, Fabrizio 

PIM1 oncoprotein is destabilized by ribosomal stress and inhibits cell cycle progression 176 

Luft, Eugenie 

Translational Control of Inducible Nitric Oxide Synthase 

by Arginine Availability and Arginase in vitro and in vivo 177 

Lukaszewicz, Maciej R. 

Phosphorothioate analogs of m7GTP are enzymatically stable inhibitors of 

cap-dependent translation 178 

Lukoszek, Radoslaw 

tRNA levels vary in Arabidopsis thaliana cells: Consequences for protein biosynthesis 179 

Manickam, Nandini 

Genetic analysis of translational accuracy 180 

MARIS, Christophe 

Structural investigation of IRES RNA stemloop H of EMCV virus in complex with PTB 

RRM1 181 

Marnef, Aline 

Pat1 and Pat2 proteins are RNA-binding proteins that repress translation in Xenopus 

laevis oocytes 182 

xxii

xxiii 

Posters A-Z 

Martinez-Salas, Encarna 

Identification of Gemin5 as a novel IRES transacting factor 183 

Mateus, Denisa 

Molecular reconstruction of a Candida genetic code alteration in Saccharomyces 

cerevisiae 184 

Mauxion, Fabienne H. 

Control of mRNA deadenylation by BTG/Tob factors 185 

Mc Mahon, Robert 

The role of host cell signaling and eIF4F in reactivation of quiescent herpes 

simplex virus type 1 (HSV-1) 186 

Mestel, Celine 

Overexpression of eIF4E regulates tumor cell invasion largely through translational 

control of ß1 integrin mRNA 187 

Milon, Pohl 

Kinetic mechanisms on mRNA selection by the ribosome 188 

Miska, Eric A. 

LIN-28 and the poly(U) polymerase PUP-2 regulate 

let-7 microRNA processing in Caenorhabditis elegans 189 

Mohammad-Qureshi, Sarah 

Characterisation of Phosphoresidues within the Catalytic Subcomplex of eIF2B 190 

Mokrejš, Martin 

Deciphering the transcriptome of all eIF4E class I, II and III genes from full-length 

cDNA, EST, HTC data from most organisms 191 

Moretti, Francesca 

Positional effects of microRNA-mediated translational regulation and their 

mechanistic basis 192 

Müller, Christine 

Nucleolar retention of a translational C/EBPalpha isoform stimulates rDNA transcription 

and cell growth 193 

Munzarova, Vanda 

Mutational analysis of the interaction between the N-terminal domain of eIF3a and the 5’ 

enhancer of uORF1 from the GCN4 mRNA leader that is critically required for efficient 

reinitiation 194 

Musner, Nicolo' 

Analysis of the stress transducer, PERK, in sciatic nerves of the CMT 1B neuropathy 

mouse 195



Musnier, Astrid 

Developmental regulation of p70 S6 kinase by a G protein-coupled 

receptor dynamically modelized in primary cells 196 

Naarmann, Isabel S. 

DDX6 is a novel regulator of reticulocyte 15-lipoxgenase mRNA translation 197 

Nadera, Ainaoui 

FGF1 induction in myogenesis depends on IRES novel cross-talks with promoter and 

3’UTR elements 198 

Nanda, Jagpreet S. 

eIF1 controls multiple steps in start codon recognition during eukaryotic translation 

initiation 199 

Napthine, Sawsan 

Translation termination – reinitiation in murine norovirus 200 

Naveau, Marie 

Role of e/aIF2 subunits in initiator tRNA binding 201 

Niedzwiecka, Anna 

Molecular mechanism of the mRNA 5’ cap binding by poly(A)-specific 3’ ribonuclease 

(PARN) 202 

Niepmann, Michael 

Influence of the Hepatitis C Virus 3´-untranslated region on IRES-dependent and 

IRES-independent translation initiation 203 

Nieradka, Andrzej 

Interaction between Grsf1 and elements within alternative exon of SCF sensitive gene – 

Use1 affects translation 204 

Niessing, Dierk 

An Extended RNA-binding Surface of She2p Oligomers is Required 

for mRNP Assembly and Localization 205 

Nikolic, Emily 

Programmed ribosomal frameshifting: a structural approach 206 

Ott, Martin 

Mrpl36 is important for generation of assembly competent proteins during 

mitochondrial translation 207 

Perez-Martinez, Xochitl 

Pet309 is a multidomain protein involved in translational activation 

of the COX1 mRNA in Saccharomyces cerevisiae mitochondria 208 

Phillips, Nicola M. 

Single Molecule Investigations of eukaryotic Initiation Factor 4A 209 

xxiv

xxv 

Posters A-Z 

Pierre, Philippe D. 

Investigating the role of translation regulation in dendritic cells 210 

Pilotte, Julie 

Expression levels of the cold-stress inducible RNA-binding motif 

3 protein, RBM3, alter the outcome of microRNA processing 211 

Pinheiro, Hugo 

Allele Specific CDH1 Down-Regulation Increases Susceptibility to Diffuse Gastric Cancer212 

Plank, Terra-Dawn 

Identification of RNA binding proteins to the HIV-1 5’ leader: insights into mechanisms of 

translation initiation 213 

Powell, Michael L. 

Further characterisation of the translational termination-reinitiation 

signal of influenza B segment 7 214 

Poyry, Tuija A. 

The mechanism of internal ribosome entry and initiation site selection 

on the FMDV IRES 215 

Prestele, Martin 


mitochondrial translation 216 

Prizant, Maya 

Cytoskeletal Control of c-Jun Translation 217 

Quattrone, Alessandro 

Widespread uncoupling between transcriptional and translational 

control of gene expression in mammalian cells 218 

Ramalho, Anabela S. 

A cystic fibrosis mutation that unravels alternative translation initiation:structural, 

functional and clinical implications 219 

Remme, Jaanus 

Ribosome reactivation by replacement of damaged proteins 220 

Ribeiro, Luís 

A new function for the Contactin associated protein 1, Caspr 1, in the regulation 

of GluR1 mRNA stability 221 

Ricci, Emiliano P. 

Untreated rabbit – reticulocyte lysate as an in vitro system to recapitulate translation 

inhibition driven by endogenous miRNAs as well as pre-miRNA processing 222 

Ricciardi, Adele S. 

Identification of SLIP1 Binding Proteins 223



Ruggeri, Valentina 

eIF6 haploinsufficiency suppresses Myc oncogenic activity 224 

Ruggero, Davide 

Genetic dissection of oncogenic PI3K-Akt-mTOR pathway reveals deregulations in 

translational control via 4EBP1-eIF4E as a 

key determinant of cancer 225 

Sandikci, Arzu 

Coordination of N-terminal enzymatic processing with 

co-translational folding and targeting of nascent polypeptides 226 

Sargueil, Bruno 

Molecular mechanisms for HIV genomic RNA translation initiation 227 

Sattlegger, Evelyn 

Gcn1 and actin binding to the central region of Yih1: Implications on 

Gcn2 activation 228 

Scheckel, Claudia 

The STAR-domain protein GLD-1 stabilizes mRNAs in the C. elegans germ line 229 

Schenk, Luca d. 

Global Identification of Potential RNA Targets for the 

Paralogous La-related RNA binding Proteins Slf1p and Sro9p 230 

Scheper, Gert C. 

Mutations in the genes encoding the eIF2B subunits lead to abnormal maturation and 

function of glial cells in the white matter of the brain 231 

Schmeing, T. Martin 

Structural Studies of Translation Initiation 232 

Schreiner, Eduard 

Mechanistic insights into quality control by the ribosome 233 

Shchepetilnikov, Mikhail 

The role of TOR kinase in regulation of translation reinitiation events 

during CaMV infection 234 

Shin, Nara 

Premature Termination Codon Containing mRNAs Are Translationally Repressed 

NMD-inhinition 235 

Shirokikh, Nikolay E. 

Quantitative analysis of ribosome-mRNA complexes at different translation stages 236 

Simmonds, Rachel E. 

Gene specific translational control by an immunosuppressive mycobacterial virulence 

factor 237 

xxvi

xxvii 

Posters A-Z 

Simonetti, Angelita 

Structural analysis of the translation initiation process 238 

Sokabe, Masaaki 

Formation of human Multi-Factor Complex with purified components 

for EM structural analysis 239 

Soppa, Joerg 

Initiation and regulation of translation in halophilic Archaea 240 

Soto Rifo, Ricardo 

A deep comparison between HIV-1 and HIV-2 reveals strong 

differences in genomic RNA localization and their translational properties 241 

Squires, Jeffrey 

Mapping 5-methylcytosine in RNA using bisulfite sequencing 242 

Stevenson, Abigail L. 

Intra-ribosome FRET and cryo-EM reveal conformational changes 

in the yeast 43S ribosomal complex 243 

Stöhr, Nadine 

ZBP1 controls mRNA turnover during stress independent of 

stress granules 244 

Susmitha, Suresh 

Characterization of ribosomal/ribosomal associated proteins 

in S.cerevisiae 245 

Szczepaniak, Sylwia A. 

Cap analog modified enzymatically stable affinity resins – 

a new tool for the analysis of cap binding proteins 246 

Tahiri-Alaoui, Abdessamad 

The 5' Leader of an immediate-early transcript from Marek's 

Disease Virus contains intronic IRES with allosteric properties 247 

Tellam, Judy T. 

Regulation of Protein Translation through mRNA Structure Influences 

MHC Class I Loading and T cell Recognition 248 

Temme, Claudia 

The Drosophila CCR4 NOT deadenylase: composition and function 249 

Terenin, Ilya M. 

Delay in eIF5-mediated hydrolysis of eIF2-bound GTP regulates 

start codon selection during translation initiation in mammals 250 

Thermann, Rolf 

Drosophila miR2 primarily targets the m7GpppN cap structure 

for translational repression 251



Thompson, Mary K. 

Ribosome-profiling of a RACK1 ribosome-binding defective mutant 

in response to Calcofluor-white induced stress 252 

Thompson, Sunnie R. 

A ribosomal protein that is essential for IRES-mediated translation 253 

Toledo, Juliano S. 

Leishmania mutants overexpressing the Spliced Leader RNA present an altered pattern 

of gene expression and are unable to 

cause infection in vivo 254 

Trabuco, Leonardo G. 

Computational studies of regulatory nascent chain recognition 

by the ribosome 255 

Tsai, Becky p. 

Identifying LEF1 IRES Protein Complexes by Mass Spectrometry 256 

Tseng, Joseph Ta-Chien 

Translational up-regulation of Aurora-A in EGFR-overexpressed cancer 257 

Tuller, Tamir 

A universal translation efficiency profile of proteins 258 

Tumer, Nilgun E. 

Ricin A chain interacts with isolated ribosomal stalk in a single 

step binding model 259 

Umesh, Varshney 

Impact of rRNA methylations on ribosome recycling and fidelity of 

initiation in Escherichia coli 260 

Valasek, Leos 

The indispensable N-terminal half of eIF3j co-operates with its structurally conserved 

binding partner eIF3b-RRM and eIF1A in stringent AUG selection 261 

Vazquez-Laslop, Nora 

Regulation of translation by the nascent peptide 262 

Veo, Bethany L. 

Identification of potential ITAFS that regulate the TAU IRES 263 

Viero, Gabriella 

The shapes of native mammalian polysomes 264 

Volta, Viviana 

Just a Minute? 265 

xxviii

xxix 

Posters A-Z 

Vopalensky, Vaclav 

Capping enzyme encoded by Kluyveromyces lactis linear plasmids doesn’t support 

cap-dependent translation of their mRNAs 266 

Wallace, Adam 

Nematode Trans-Spliced Leader Sequence and Structure Required for Translation of 

TMG-Capped mRNAs 267 

Weil, Dominique 

Unravelling the ultrastructure of stress granules and associated P-bodies 

in human cells 268 

Wie, Sten m. 

Translational Regulation of the Thymidylate Synthase mRNA 269 

Wieden, Hans-Joachim 

Emerging roles of the universally conserved GTPases HflX and YchF during protein 

synthesis 270 

Wilhelm, Jim 

The eIF4E binding portein Hubcap defines a novel class of localized RNPs 271 

Willett, Mark 

Translational Control during Cell Spreading, Adhesion and Migration 272 

Willis, Anne E. 

Polypyrimidine tract binding protein is a regulator of cytoskeletal organisation 

and cell migration 273 

Wolf, Annika 

Plakophilin 1 stimulates cell proliferation and growth by promoting eIF4A activity 274 

Woo, Connie Wai Hong 

Prolonged Physiologic ER Stress Triggers Adaptive Suppression of ATF4-CHOP by a 

Mechanism that Appears to "Compensate" for the Protein Translational Effects of P-eIF2 

alpha 275 

Wypijewska, Anna 

Substrate specificity of C. elegans scavenger decapping enzyme DcpS for m7GpppG, 

m2,2,7GpppG and chemically modified dinucleotide cap analogues 276 

Yanagiya, Akiko 

Charactarization of the role of PABP interacting protein 2 in the late stage of 

spermatogenesis in PAIP2 knockout mouse model 277 

Yángüez, Emilio 

Influenza virus requirements for eIF4F components: hijacking cellular translation 

machinery 278 

Yatime, Laure 

Structure of the RACK1 dimer from Saccharomyces cerevisiae 279



Young, Lucy A. 

Translational reprogramming following DNA damage 280 

Zaborowska, Izabela 

A role for Vaccinia Virus I3L in the redistribution of host translation factors to cytosolic 

viral replication compartments 281 

Zhang, Yonglong 

Characterization of YafO, an Escherichia coli toxin 282 

Zilow, Sonja 

Upstream of N-Ras (Unr) is involved in Translational Control of ADAM10 283 

Zuberek, Joanna 

Binding specificities of multiple Drosophila eIF4E family members to cap structure 284 

xxx

JUDITH KIMBLE 

1 

Speaker Abstracts 

Translational control and stem cells: lessons from the C. elegans germline 

University of Wisconsin-Madison, United States of America 

Stem cells maintain themselves by self-renewal and also produce differentiated cells. My 

laboratory investigates stem cell controls in the experimentally tractable nematode 

Caenorhabditis elegans with its cellular simplicity and powerful genetics/genomics. In this 

small worm, a mesenchymal stem cell niche employs Notch signaling and a downstream RNA 

regulatory network to control the germline decision between self-renewal and differentiation. 

Two key nodes in this network are FBF and GLD-2. FBF is a PUF (for Pumilio and FBF) 

RNA-binding protein that is required for stem cell maintenance; GLD-2 is the catalytic subunit 

of a cytoplasmic poly(A) polymerase that activates differentiation. I will discuss recent findings 

that identify conserved PUF target mRNAs, advance our understanding of FBF control 

mechanisms and reveal distinct GLD-2 partners for antagonistic fates. Our findings expand the 

C. elegans RNA regulatory network controlling stem cells and their progeny, and have 

important implications for understanding RNA controls more broadly.


EMBL Heidelberg, 9-13 September 2009 

DAVID BARTEL 

MicroRNAs 

MIT/Whitehead Institute/HHMI, United States of America 

MicroRNAs are small endogenous RNAs that can guide the posttranscriptional repression of 

protein-coding genes. We have been using molecular and computational approaches to find 

microRNAs in plants and animals, identify the messages that they repress, and then investigate 

their functions during development, oncogenesis, and other processes. This talk will focus on 

recent results shedding light on the genomics of metazoan microRNAs and their regulatory 

targets, including analyses of high-throughput sequencing of small RNAs, computational and 

experimental results revealing the widespread impact of miRNAs on mRNA evolution and 

protein output, and methods for identifying mRNAs that are most effectively repressed by 

miRNAs. 

2

REINHARD LUEHRMANN 

At the heart of the spliceosome 

MPI fuer Biophysikalische Chemie, Germany 

3 


The spliceosome is a multi-MDa RNP machine that consists of the small nuclear (sn)RNPs U1, 

U2, U4/U6 and U5, and numerous non-snRNP proteins. The spliceosome exhibits unique 

design principles that are unprecedented among RNP machines. For example, the stepwise 

interaction of the snRNPs with the pre-mRNA during spliceosome assembly culminates with 

the formation of the so-called B complex which still lacks an active site. During the subsequent 

catalytic activation step major RNA-RNA and RNP remodelling events occur, generating the 

activated B* complex, which then catalyses the first step of splicing to yield the C complex. To 

investigate biochemically the transitions from the pre-catalytic B complex to B* and C 

complexes, we affinity purified B, B* and C complexes under native conditions from human, D. 

melanogaster and S. cerevisiae. Proteomic analyses of these complexes revealed that the 

dramatic compositional dynamics during catalytic activation are conserved across evolution, 

but that the yeast spliceosome has a radically lower number of proteins compared to 

metazoans, and thus its proteome represents the conserved core set of proteins required for 

splicing. We purified human C complexes that exhibit catalytic activity on their own and defined 

their stable RNP core. Moreover, we established an in vitro splicing complementation system 

that allows to reconstitute both steps of yeast splicing with purified components. I will also 

report on recent advances in 3D structural studies of human and yeast spliceosomes using 

electron cryomicroscopy and in localizing important proteins and pre-mRNA sequences within 

spliceosomes by immuno-EM. Finally, I will present crystal structures of several functionally 

important proteins of the U4/U6.U5 tri-snRNP including Prp8 and Brr2.



JON LORSCH 

Reconstitution and Analysis of Cap-stimulated mRNA Recruitment to the 43S 

Pre-initiation Complex 

Johns Hopkins University School of Medicine, United States of America 

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we 

have developed a reconstituted translation initiation system using S. cerevisiae components. 

This system initially used an unstructured, model mRNA to remove the need for factors 

involved in mRNA recruitment. We have now expanded the system to include 5'-cap 

stimulated mRNA loading and analyzed the factor requirements of this important step. Our 

data indicate that eIF3 alone is sufficient for maximal recruitment of unstructured, model mRNA 

to the pre-initiation complex. In contrast, natural mRNA requires eIF3 as well as eIF4F (eIF4F = 

eIF4E, eIF4G, eIF4A) and, surprisingly, eIF4B. eIF4B, a ssRNA binding protein, has generally 

been considered an accessory factor to the DEAD-box helicase eIF4A rather than a central 

player in its own right. Yeast eIF4B consists of an N-terminal RRM domain and a C-terminal 

domain (CTD) of 7 sequence repeats. We have demonstrated that eIF4B binds specifically to 

the 40S subunit in addition to ssRNA. Point mutations in the RRM have identified it as being of 

primary importance in ssRNA binding, while CTD deletions have implicated the C-terminal 

region in 40S subunit binding. Deletion of 6 of the 7 repeat sequences in the CTD severely 

inhibited the activity of eIF4B in mRNA recruitment to the pre-initiation complex in vitro, while 

mutations in the RRM domain that abrogate ssRNA binding had no effect. Addition of a 

second repeat restores 40S binding and increases mRNA recruitment activity. These data are 

consistent with in vivo work that demonstrated the importance of the C-terminal region of yeast 

eIF4B in complementation of the slow growth phenotype of the eIF4B deletion strain1. Our 

results suggest that eIF4B does not serve solely as an accessory ssRNA binding protein but 

instead plays a central role in mRNA recruitment that involves its interaction with the 40S 

ribosomal subunit. 

[1] Niederberger et al., (1998) RNA, 4, 1259-1267. 

4

ADESH SAINI 

5 


Structural elements in eIF1A regulate AUG selection by controlling distinct 

modes of initiator binding to the preinition complex 

Jagpreet Nanda 1, Jon Lorsch 1, Alan Hinnebusch 2, Adesh Saini 2 

1 Johns Hopkins School of Medicine, United States of America 

2 National Institute of Health, United States of America 

eIF1A is the eukaryotic ortholog of bacterial initiation factor 1(IF1), but contains an additional 

helical domain and long unstructured N- and C-terminal tails (NTT and CTT). We identified a 

repeated motif in the CTT (SE-1 and SE-2) that promotes recruitment of the 

eIF2·GTP·Met-tRNAiMet ternary complex (TC), and also suppresses initiation at non-AUG 

codons. Compound mutations affecting both SEs produce stronger defects in TC recruitment 

in vitro and in vivo (Gcd- phenotype), greater UUG initiation in vivo (Sui- phenotype), and 

stronger growth defects compared to mutating SEs individually, and the complete elimination 

of both SEs is lethal. Remarkably, the elevated UUG initiation and growth phenotypes of SE 

mutations are suppressed by overexpressing eIF1, a putative scanning enhancer, and by 

mutations in three other segments of eIF1A: the NTT (SI-1) and the structured N- and C- 

strands that pack against α2 in the helical domain (SI-2N and SI-2C). Strikingly, SI mutations 

also rescue the TC binding/Gcd- defects conferred by SE mutations and a Sui-/Gcd- mutation 

in eIF2β. These results indicate that SE and SI elements regulate start codon selection through 

opposing effects on TC binding. We envision that TC binds to the scanning-conducive 

conformation of the preinitiation complex in a manner that prevents base-pairing between 

initiator and P-site triplets (P[out] state), and that the transition to a scanning-arrested 

conformation is promoted by perfect codon-anticodon pairing at AUG (P[in] state). SE 

elements appear to stabilize TC binding in the P[out] state and thereby promote scanning and 

suppress non-AUG initiation, whereas SI elements antagonize TC binding in the P[out] state to 

enhance P[in] binding and start codon recognition. Biochemical analysis indicates that SE 

elements also stimulate eIF1 dissociation specifically at AUG codons, and loss of this function 

likely further enhances non-AUG initiation in SE mutants.



MARTIN JENNINGS 

eIF5 acts as a GDP dissociation inhibitor in addition to its GAP function 

Martin Jennings, Graham Pavitt 


During protein translation, the G-protein eIF2 is activated by interaction with GTP. In this GTP 

bound state, eIF2 interacts with tRNAiMet to form ternary complex before interacting with the 

40S ribosomal subunit. During AUG start codon recognition, eIF2-bound GTP hydrolysis is 

activated by the GTPase activating protein (GAP) eIF5. eIF2·GDP is then reactivated by the 

guanine nucleotide exchange factor (GEF) eIF2B to facilitate continuous translation. 

Recent work characterised a large cellular pool of an eIF5/eIF2 complex lacking tRNAiMet and 

genetic evidence suggested this complex may antagonise nucleotide exchange by eIF2B. eIF5 

has a conserved N-terminal domain (NTD) providing GAP activity, and C-terminal domain 

(CTD) critical for interactions with several initiation factors. These are joined by a linker region 

(LR). We investigated the function of this eIF2/eIF5 complex. By monitoring [3H]-GDP 

dissociation from eIF2 we have demonstrated that eIF5 acts as a guanine nucleotide 

dissociation inhibitor (GDI), i.e. eIF5 inhibits the spontaneous release of GDP from eIF2. 

A range of eIF5 mutants were used to further characterise this novel activity. These 

experiments demonstrate that the GDI and GAP functions are separate and confirm the CTD is 

important for interaction with eIF2. Furthermore, the LR is demonstrated to interact with eIF2g 

through evolutionary conserved residues and this interaction is shown to be required for GDI 

activity 

We propose that eIF2•GDP is released from the ribosome in complex with eIF5, preventing 

premature/spontaneous release of GDP from eIF2. This would ensure that eIF2B GEF activity 

is rate limiting, enabling tight control of translation initiation. In support of this, our in vivo 

evidence shows both eIF5 CTD & LR are important for sensitivity to eIF2(aP) and induction of 

GCN4. 

6

AKIRA FUKAO 

Enhancement of cap-dependent translation by the ELAV protein HuD: 

A novel function of HuD which is eIF4A- and poly(A)-dependent 

7 


Akira Fukao 1, Kunio Inoue 1, Hiroshi Sakamoto 1, Yumi Sasano 1, Toshinobu Fujiwara 1, 

Nahum Sonenberg 2, Christian Thoma 3, Hiroaki Imataka 4 

1 Kobe University, Japan 

2 McGill University, Canada 

3 University Hospital of Freiburg, Germany 

4 University of Hyogo, Japan 

RNA-binding proteins have emerged as specific regulators of gene expression. The 

RNA-binding protein HuD, one member of the neuronal Hu family of proteins promotes 

neuronal differentiation by an unknown mechanism. Here we show that HuD enhances 

translation. Translation stimulation by HuD requires both a poly(A) tail at the 3´ end of mRNAs 

and a 5´ m7G cap structure. We also show that HuD associates with the cap-binding complex 

via direct binding to eIF4A and poly(A) and that the poly(A) and eIF4A binding activity of HuD 

are critical for its translational enhancer function because HuD-eIF4A- and HuD-poly(A) binding 

mutants fail to stimulate translation. We also find that these molecular interactions are 

important for HuD-induced neurite outgrowth in PC12 cells. Our results reveal a novel function 

of HuD in translation, which might explain at least in part how HuD promotes the induction of 

neuronal differentiation.



FRANCK MARTIN 

Tethering of ribosomes downstream of translational start codon drives 

translation of histone H4 

Franck Martin 1, Sophie Jaeger 1, Laure Schaeffer 1, Lydia Prongidi-Fix 1, Gilbert Eriani 1, 

Sharief Barends 2 

1 “Architecture et Réactivité de l’ARN” UPR 9002 du CNRS, Université de Strasbourg, IBMC, 

15, rue René Descartes, F-67084 Strasbourg cedex, France 

2 ProteoNic, Niels Bohrweg 11-13, 2333 CA Leiden, Netherlands 

In metazoans, histones are produced exclusively during the S-phase of the cell-cycle in order 

to provide the large amounts of proteins needed for genome packing. These proteins are 

translated from non-conventional mRNA in that they are not polyadenylated at their 3’ end but 

instead end in a highly conserved hairpin structure. In addition, 5’ and 3’ UTRs are usually 

rather short. In eukaryotes, one crucial step of translation initiation consists in the binding of the 

multifactor complex eIF4F to the 5’ end of the mRNA. The cap-binding factor eIF4E binds the 

m7G cap structure, prerequisite to the subsequent recruitment of the activated small ribosomal 

43S particle. Here we show that the mRNA of histone H4 contains structural elements in the 

coding sequence that, dictate an alternative translation initiation process. The mRNA is folded 

in order to bind eIF4E without the need of the cap. From this internal binding site, ribosomal 

43S particles are recruited and directly loaded on the AUG by a tethering mechanism. In 

addition, H4 mRNA also contains a tight RNA structure that sequesters the cap and prevents 

canonical binding of eIF4E to the 5’-end of the mRNA. This unconventional translation initiation 

is consistent with the small sized 5’ UTR, seemingly too short to account for the conventional 

scanning-initiation model. 

8

RIVKA DIKSTEIN 

Characterization of TISU, a translation initiator specific to mRNAs with 

extremely short 5’UTR 


9 


Regulation of expression of protein-encoding gene occurs primarily at the initiation stage of 

transcription and translation. Transcription is controlled by regulatory elements, proximal and 

distal to the transcription start site (TSS). Some proximal elements are localized downstream to 

the TSS, and are present in the mRNA as well, often in the 5’UTR, where they could also 

influence translation. However there is little evidence for the existence of such common 

transcription and translation regulatory elements. We have identified an element downstream 

to the TSS up to position +30, which, remarkably, controls the initiation stages of both 

transcription and translation. This composite element is present in genes encoding for proteins 

involved in basic cellular functions such as respiration, protein metabolism and RNA synthesis. 

We showed this element to be essential for transcription initiation and promoter strength. The 

core of the motif has an invariable ATG sequence, which serves also as the translation initiation 

codon in the majority of the genes. In these genes the initiating AUG is preceded by an 

unusually short 5’UTR. We demonstrated that translation in vitro and in vivo is initiated 

exclusively from the AUG of this motif, and that the AUG flanking sequences create a strong 

translation initiation context that is distinguished from the well-known Kozak in its unique ability 

to direct efficient and accurate translation initiation from mRNAs with a very short 5’UTR 

without ribosome scanning. We therefore named it TISU for Translation Initiator of Short 

5’UTR. Our findings suggest that the transcription and translation processes, once considered 

to be independent of each other due to cellular compartmentalization, are linked through a 

common regulatory element.



CHRISTINE LUTTERMANN 

The importance of inter- and intramolecular base pairing for translation 

reinitiation on a eukaryotic bicistronic mRNA 

Christine Luttermann, Gregor Meyers 

Friedrich-Loeffler-Institut, Germany 

Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping 

cistrons. The first open reading frame of this RNA codes for the viral major capsid protein VP1 

and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a 

termination/reinitiation mechanism, which depends on an upstream sequence element of 

about 70 nucleotides denoted “termination upstream ribosomal binding site” (TURBS). Two 

short sequence motifs within the TURBS were found to be essential for reinitiation. Motif 1 is 

conserved among caliciviruses and is complementary to a region within helix 26 of mammalian 

18S rRNA. By a whole set of single site mutations and reciprocal base exchanges we 

demonstrate for the first time conclusive evidence for the necessity of mRNA/18S rRNA 

hybridization for translation reinitiation in a eukaryotic system. Moreover, we show that motif 2 

exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus 

forming a secondary structure that positions post-termination ribosomes in an optimal distance 

to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better 

understanding of the requirements for translation termination/reinitiation in eukaryotes. 

10

ANNA MARIA GIULIODORI 

Translational regulation of cold-shock gene expression 

11 


Cynthia Pon, Mara Giangrossi, Anna Brandi, Anna Maria Giuliodori, Claudio Gualerzi 

University of Camerino, Italy 

All living organisms have evolved mechanisms to cope with the effects caused by sudden 

temperature downshifts (cold-shock). Following cold-shock Escherichia coli enters an 

acclimation phase during which cell growth stops for 3-6 hours, bulk gene expression is 

drastically reduced and the expression of a set of cold-shock genes is selectively and 

transiently enhanced. 

Cold-shock gene expression is mainly subject to translational control, promoted by both 

cis-acting elements of the transcripts and trans-acting factors, whose levels increase during 

cold-shock. To elucidate the nature of these cis-acting elements, we determined the 

secondary structure of cspA mRNA, encoding for CspA, the best studied cs protein in E. coli. 

This allowed us to demonstrate that cspA mRNA undergoes a temperature-dependent 

structural rearrangement, likely resulting from stabilization in the cold of an otherwise 

thermodynamically unstable transcriptional intermediate, thereby acting as a thermosensor. 

Unlike the 37°C-structure, the cold–shock structure of cspA mRNA is endowed with a rather 

open conformation of the translation initiation region, which favours its translation. In turn, the 

accumulation of CspA stimulates translation of its own as well as of other mRNAs at low 

temperature. Other important trans-acting factors involved in the cold-shock translational 

regulation are IF3 and IF1, whose levels with respect to ribosomes increase considerably 

during cold shock. The increased IF3-IF1/ribosome ratio is essential to overcome the higher 

stability of 70S monomers at low temperature so as to provide a sufficient pool of dissociated 

30S subunits capable of ‘‘70S initiation complex’’ formation. Furthermore, IF3 is able to 

stimulate selectively, at low temperature and in a dose-dependent manner, translation of 

cold-shock mRNAs, while discriminating against non-cold-shock mRNAs causing them to form 

non-productive 70S initiation complexes.



DIETER WOLF 

The eIF3 interactome reveals the translasome, a supercomplex linking protein 

synthesis and degradation machineries 

Zhe Sha 1, Eric C. Chang 1, Rodrigo Cabrera 1, Dieter Wolf 2, Laurence M. Brill 2, Michael H. 

Glickman 3, Oded Kleifeld 3 

1 Baylor College of Medicine, United States of America 

2 Burnham Institute for Medical Research, United States of America 

3 Technion - Israel Institute of Technology, Israel 

eIF3 promotes initiation of translation in vitro, but relatively little is known about how it 

accomplishes its function in vivo. Here, we employed affinity purification and highly sensitive 

LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 

proteins. eIF3 assembles into a large supercomplex, the translasome, which contains 

elongation factors, tRNA-synthetases, 80S ribosomes, chaperones, and the proteasome. eIF3 

also associates with ribosome biogenesis factors and the b-importins Kap123p and Sal3p, 

suggesting that it has nuclear functions. Genetic data indicated that the binding of 

beta-importins is essential for cell growth, and photobleaching experiments revealed a critical 

role for Sal3p in the nuclear import of one of the translasome constituents, the proteasome. 

Our data revealed an unexpected breadth of the eIF3 interactome and suggest that molecules 

involved in translation initiation, ribosome biogenesis, translation elongation, quality control, 

and transport are physically linked to allow for efficient protein synthesis. 

12

HELENA FIRCZUK 

13 


Comprehensive rate control analysis of the eukaryotic translation pathway 

John E.G. McCarthy, Helena Firczuk, Shichina Kannambath, Xi Wang, Helen Bryant, Hans 

Westerhoff, Pedro Mendes 

Manchester Interdisciplinary Biocentre, United Kingdom 

Elucidation of the control principles governing a complex pathway like translation can only be 

achieved via quantitative systems analysis. We have performed the first comprehensive in vivo 

rate control analysis of the yeast translation machinery. Using the tet07 operator we have 

progressively suppressed transcription of each translation factor gene and have determined 

how this affects yeast growth rate and in vivo protein synthesis rate. The relationship between 

the protein synthesis rate and cellular concentration of each factor enables us to estimate the 

sensitivity coefficient CJ. The latter reflects how each individual translation factor contributes to 

the steady state translation rate. For most of the proteins we also explored the CJ values at 

cellular concentrations above wt levels (>100%). This in vivo analysis has been extended by 

parallel in vitro experiments in a yeast cell-free system, allowing us to assess the effects of 

ribosome:mRNA saturation on control. All three stages of translation (initiation, elongation and 

termination) have been represented as a set of ordinary (and/or stochastic) differential 

equations and simulated using the software COPASI. 

Translational control is distributed over multiple components of the translation machinery. 

Some components, for example eIF1, eIF3a and eIF4A, show strong control (large CJ). Others, 

for example Pab1 and eIF5, show surprisingly low CJ values – indeed, these factors appear to 

be present at levels in excess (~50%) to the requirements of translation. Generally, the value of 

CJ shows no simple correlation with a factor’s intracellular concentration. These, and other, 

results reveal many unexpected features of the system that could not have been predicted on 

the basis of non-quantitative data.



NICHOLAS INGOLIA 

Genome-wide analysis of in vivo translation with single-nucleotide resolution 

Nicholas Ingolia, Jonathan Weissman 

University of California, San Francisco, United States of America 

We have developed a technique, called ribosome profiling, that enables genome-wide 

investigation of translation with single-nucleotide resolution. Our approach is based on 

nuclease footprinting of in vivo derived translating ribosomes followed by deep sequencing of 

ribosome-protected mRNA fragments. In the course of developing this technique, we 

optimized a broadly applicable protocol for generating deep sequencing libraries from small 

RNA fragments. 

We used ribosome profiling to comprehensively monitor translation in budding yeast. Our 

studies defined the exact protein sequences being translated, including both their position on 

the mRNA and the reading frame being decoded. We also quantified translation with high 

precision and measured differences in translational efficiency between different messages that 

play a major role in determining absolute protein abundance. We further measured translational 

regulation by using ribosome profiling on cells subject to amino acid starvation, an 

environmental stress which is known to affect translation. 

Ribosome profiling revealed novel features of translation, including the existence of distinct 

phases characterized by a large decrease in ribosome density going from early to later peptide 

elongation. In addition to translation of protein-coding genes, we observed translation of 

upstream open reading frames as well as wide-spread initiation at non-AUG codons. Upstream 

non-AUG initiation changed in response to starvation and may play a role in regulating the 

translation of downstream protein-coding genes. 

We have adapted ribosome profiling to mammalian cells and present preliminary 

measurements of in vivo translation in higher eukaryotes. 

14

TERRI KINZY 

15 


ADP-ribosylation of eukaryotic elongation factor 2 by bacterial toxins and its 

effects on translation elongation in vivo and in vitro 

Maria M. Mateyak 1, Pedro Ortiz 1, Eric Jan 2, Terri Kinzy 3 

1 Department of Molecular Genetics, Microbiology, and Immunology, Robert Wood Johnson 

Medical School, UMDNJ, Piscataway, NJ 08854, United States of America 

2 Department of Biochemistry and Molecular Biology, University of British Columbia, 

Vancouver, BC V6T 1Z3, United States of America 

3 UMDNJ Robert Wood Johnson Medical School, United States of America 

ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) by bacterial toxins leads to inhibition 

of its function in translation elongation. Diphtheria toxin, produced by Corynebacterium 

diphtheria, and exotoxin A, produced by Pseudomonas aeruginosa, ADP-ribosylate eEF2 on a 

modified histidine residue at position 699 called diphthamide, that is highly conserved 

throughout eukaryotes. However, mechanistically how ADP-ribosylation of eEF2 inhibits its 

function is poorly understood. We have developed a novel Saccharomyces cerevisiae system 

to directly address the effects of ADP-ribosylation on eEF2’s role in translation elongation in 

vivo. This system employs eEF2 mutations identified within the anticodon mimicry domain that 

display dominant resistance to galactose-induced diphtheria toxin expression in yeast. These 

eEF2 mutants lack the diphthamide modification and are not a target of diphtheria toxin; 

however, their expression allows growth in the presence of ADP-ribosylated wild type eEF2, 

thus providing an excellent system to study the in vivo effects of eEF2 ADP-ribosylation. For 

example, yeast expressing both mutant and wild type eEF2 display an increase in parmomycin 

sensitivity specifically upon diphtheria toxin induction demonstrating a link between 

ADP-ribosylation of eEF2 and translation defects in vivo. These effects are due to the 

presence of the ADP-ribosylated form of eEF2, as determined by MS analysis. Interestingly, 

the level of ADP ribosylation and the presence of a pool of eEF2 only partially modified at 

histidine 699 to diphthamide and thus resistant to ADP ribosylation in vivo is not consistent 

with a complete inhibition of all eEF2 activity. However, in vitro analysis indicates alterations in 

the anticodon mimicry domain, which includes the location of the ADP ribosylation target 

diphthamide 699, clearly affect translocation. In summary, the establishment of this novel yeast 

system will provide insight into this fundamental and long-standing question of the role of the 

ADP-ribosylation on eEF2



ALENA PALESKAVA 

Unusually tight binding of Sec-tRNASec to the elongation factor SelB due to 

the specific recognition of the selenocysteyl group by the GTP-bound form of 

SelB 

Alena Paleskava, Andrey L. Konevega, Marina Rodnina 

Max Planck Institute for Biophysical Chemistry, Germany 

SelB is a specialized translation elongation factor which binds selenocysteyl-tRNA 

(Sec-tRNASec) and a SECIS element of the mRNA and promotes recoding of UGA stop 

codons as selenocysteine. The aim of the present work was to understand the mechanism by 

which SelB specifically recognizes Sec-tRNASec. We show that the affinity of Sec-tRNASec 

binding to SelB•GTP is extremely high (Kd ≈ 0.2 pM) and is more than a million-fold higher 

than that to the GDP-bound or the apo forms of the factor, despite the similar structures of 

SelB in the GTP-, GDP-bound and apo forms. The tight binding of Sec-tRNASec to SelB•GTP 

correlates with the net formation of four ion pairs, three of which seem to involve the Sec 

residue. The high selectivity for SelB•GTP is restricted to Sec-tRNASec, whereas Ser-tRNASec 

and deacylated tRNASec bind to all forms of SelB with equally low affinity (Kd ≈ 0.2 μM). 

Furthermore, while the SelB•GTP•Sec-tRNASec complex is very stable kinetically, with a 

half-life time in the hours range, GTP hydrolysis increases the dissociation rate constant by six 

orders of magnitude, to about 200 s-1. The destabilization of Sec-tRNASec interactions with 

SelB explains why GTP hydrolysis is required for the release of Sec-tRNASec from SelB upon 

delivery of Sec-tRNASec to the ribosome. The tRNA-binding properties of SelB are reminiscent 

of those of another specialized factor, eukaryotic translation initiation factor eIF2, rather than 

those of EF-Tu, the common delivery factor for all other aminoacyl-tRNAs, supporting the idea 

of a common evolutionary origin of SelB and eIF2. In contrast to EF-Tu, which binds all 

elongator aminoacyl-tRNAs with uniform affinity, recognition by SelB and eIF2 of their 

respective aminoacyl-tRNAs has evolved to sense the amino acid moiety of aa-tRNA. 

16

JAMES MUNRO 

17 


Single-molecule observations of rate-limiting conformational events during 

ribosomal translocation 

Kevin Sanbonmatsu 1, Chang-Shung Tung 1, Jamie Cate 2, James Munro 3, Scott Blanchard 3, 

Roger Altman 3, Michael Wasserman 3 

1 Los Alamos National Laboratory, United States of America 

2 UC Berkeley, United States of America 

3 Weill Cornell Medical College, United States of America 

The mechanism by which tRNA and mRNA translocate with respect to the ribosome is critical 

to gene expression. Contemporary models posit a multi-step, kinetically-driven translocation 

mechanism, rate-limited by conformational processes in the ribosome and elongation factor G 

(EF-G). However, the precise order and timing and specific roles of distinct conformational 

degrees of freedom in the ribosome complex remain open questions that have thus far been 

difficult or impossible to delineate. Here, using single-molecule fluorescence resonance energy 

transfer (smFRET), we have investigated the order and timing of conformational changes in the 

ribosome complex critical to tRNA-mRNA movements. By obtaining data from multiple 

structural perspectives we have directly monitored the events leading up to formation of the 

unlocked ribosome conformation, in which the P/E hybrid state is formed, the small ribosomal 

subunit is ratcheted, and the L1 stalk has adopted a closed conformation. These results reveal 

that conformational processes on path to the unlocked state do not occur in a concerted 

fashion, but are instead loosely coupled. Under equilibrium conditions, tRNAs exchange 

between hybrid and classical states rapidly, subunit ratcheting-unratcheting exchange occurs 

relatively slowly, and unlocked state formation is rate-limited by L1 stalk closure. The loosely 

coupled nature of these events specifies that multiple structural pathways may be transited 

during translocation. Direct observations of EF-G binding and translocation events suggest that 

EF-G captures the unlocked state of the ribosome, inhibiting its return to the locked 

configuration and promoting a fast dynamic mode of the L1 stalk. In this model, opening of the 

L1 stalk on the ratcheted/P/E hybrid state ribosome contributes directly to movement of P-site 

tRNA to the E site.



SHASHI BHUSHAN 

Visualization of nascent chains in the ribosomal exit tunnel: Implication for 

secondary structure formation 

Shashi Bhushan 1,2 , Marco Gartmann 1,2 , Mario Halic 1,2 , Jean-Paul Armache 1,2 , Alexander 

Jarasch 1,2 , Thorsten Mielke 3 , Daniel N. Wilson 1,2 , Roland Beckmann 1,2 

1 Gene Center and Department for Chemistry and Biochemistry, University of Munich, 

Feodor-Lynen-Str. 25, 81377 Munich, Germany 

2 Center for integrated Protein Science Munich (CiPSM), University of Munich, 81377 Munich, 

Germany 

3 UltraStrukturNetzwerk, Max Planck Institute for Molecular Genetics, Ihnestr. 73, 14195-Berlin, 

Germany 

As translation proceeds, the nascent polypeptide chain passes through a tunnel in the large 

ribosomal subunit. Once thought only to be a passive conduit for the growing nascent chain, 

accumulating evidence suggests that the ribosomal exit tunnel may in fact play a more active 

role in regulating translation and initial protein folding events. In order to study the possibility of 

secondary structure formation within the tunnel, three different nascent chains containing a 

either strong helix forming sequence of five EAAAK repeats, 19 consecutive alanine residues or 

a hydrophobic transmembrane helix segment (7 Leu/12 Ala) were used as a substrate. 

Ribosome nascent chain complexes (RNCs) were generated using an in vitro wheat germ 

translation system and purified using affinity tags in the nascent chains. Cryo-EM and 

single-particle reconstruction of these RNCs resulted in the direct visualization of the substrate 

nascent chains, both in helical as well as in extended conformations. In addition to the direct 

visualization of density for the nascent chains, the high quality of the maps enables the sites of 

interaction with the tunnel wall components to be elucidated. In particular regions of the tunnel, 

the nascent chain is seen to adopt distinct conformations and establish specific contacts with 

tunnel components - both ribosomal RNA and proteins. 

18

C. AXEL INNIS 

Shedding Light Onto Nascent Chain-Mediated Translational Stalling 

19 


Birgit Seidelt 1, Daniel Wilson 1, Marco Gartmann 1, Jean-Paul Armache 1, Thomas Becker 1, 

Roland Beckmann 1, Thomas Steitz 2, Thorsten Mielke 3, Leonardo Trabuco 4, 

Klaus Schulten 4,C. Axel Innis 5 

1 Gene Center and Department of Chemistry and Biochemistry / CiPSM, University of 

Munich, Germany 

2 HHMI / Yale University, United States of America 

3 Max Planck Institute for Molecular Genetics, Berlin, Germany 

4 University of Illinois at Urbana-Champaign, United States of America 

5 Yale University, United States of America 

Although most nascent polypeptide chains are thought to transit passively through the exit 

tunnel of the ribosome during translation, a number of regulatory peptide sequences have 

been proposed to interact with the exit tunnel specifically, causing ribosomes to stall. One of 

the best studied examples of nascent chain-mediated translational stalling is the TnaC system, 

in which the expression of genes involved in the catabolism of soluble tryptophan is switched 

on or off in response to intracellular levels of this amino acid. This process has been shown to 

be dependent upon ribosome stalling during translation of the 24-residue TnaC leader peptide 

and the interaction between the resulting nascent chain and the ribosomal tunnel are critical for 

efficient stalling. Here, we present the 5.8 Å resolution cryo-EM and single particle 

reconstruction of an E. coli 70S ribosome stalled during translation of the tnaC leader gene. 

The high quality of the experimental map reveals how an ordered TnaC nascent chain makes 

discrete contacts with the walls of the ribosomal exit tunnel. Moreover, the universally 

conserved A2602 and U2585 at the peptidyl transferase center adopt conformations that are 

incompatible with co-habitation of the termination release factors. Put together, these 

observations allow us to propose a general model by which structural changes produced 

within the ribosome by TnaC are relayed to and inactivate the peptidyl transferase center. In 

addition, our study provides compelling evidence that nascent chains can adopt distinct 

conformations within the exit tunnel.



KRISTEN BARTOLI 

Novel Role for Mitotic Microtubule Motor Protein Eg5 in Protein Translation 

Jelena Jakovljevic 1, William Saunders 2, John Woolford 1, Kristen Bartoli 3 

1 Department of Biological Sciences, Carnegie Mellon University, United States of America 

2 Department of Biological Sciences, University of Pittsburgh, Germany 

3 University of Pittsburgh, School of Medicine, United States of America 

Kinesin motor proteins are important for the transport of specific intracellular cargo along 

microtubules. In addition to this transport function, some kinesins have been implicated in 

mitosis and are critical for proper cell division. Eg5 is a plus-end directed microtubule motor 

that localizes to spindles during mitosis. To date the characterization of Eg5 has almost 

exclusively been limited to its role in mitosis. Although recently, we have determined through 

ribosomal subunit fractionation studies and immunoprecipitation, that during interphase Eg5 

predominantly associates with ribosomes. In addition, inhibition of Eg5 function, either by a 

knockdown or a small molecule inhibitor, resulted in a marked decrease of ~50% in protein 

translation. Through polysome profiling studies, we have determined that after knockdown or 

inhibition of Eg5, the decrease in protein translation is accompanied by an increase in the 80S 

complex, which suggests a defect in translation elongation. Furthermore, through in vitro 

microtubule binding assays, we observed that the 80S ribosome binds microtubules via Eg5 in 

mammalian cells. Collectively, our data proposes that Eg5s major function in a cell is to aid 

protein translation and suggests a novel role for mitotic microtubule motor Eg5 in translation. 

20

ERIK BÖTTGER 

21 


Aminoglycoside Ototoxicity and Synthesis of New Compounds with Altered 

Drug-Target Interaction 

D. Fernandez 1, S.R. Dubbaka 1, A. Vasella 1, D. Sherbakov 2, S. Kalapala 2, A. Subramanian 2, 

T. Matt 2, M. Kulstrunk 2, Erik Böttger 2, Rashid Akbergenov 2 

1 ETH Zürich, Laboratorium für Organische Chemie, Switzerland 

2 Institut für Medizinische Mikrobiologie, Universität Zürich, Switzerland 

Aminoglycoside antibiotics interfere with ribosomal decoding by binding to the small subunit’s 

A-site. While these antibiotics preferentially target prokaryotic ribosomes, they are associated 

with significant toxicity. Recent data have identified the mitoribosome as a component 

important in aminoglycoside toxicity. The link between toxicity and aminoglycoside-induced 

malfunction of the mitochondrial ribosome prompted us to address the question whether it is 

possible to redirect drug-target interaction, so as to develop more selective aminoglycoside 

derivatives, i.e. compounds, which have lost the affinity for the mitoribosome while retaining the 

antibacterial activity. Key ribosomal structures relevant for drug binding involve the 

phylogenetically polymorphic 16S rRNA positions 1408, 1409 and 1491. We have used 

site-directed mutagenesis to generate a set of mutants characterized by single point mutations 

in the drug binding site. We used this set of mutants to guide a step-by-step synthesis of novel 

aminoglycoside compounds. Initially, we focussed our efforts on modification of residues C(4’), 

C(6’) and C(5’’) as we wished to specifically modify A1408-ring I and G1491-ring III 

interactions. 

After having synthesized various aminoglycoside derivatives with specific modifications 

introduced at the residues chosen, we were eventually able to synthesize a series of 

compounds which maximally exploit the phylogenetically diverse rRNA positions present in the 

drug binding pocket. Compared to available aminoglycosides the series of aminoglycosides 

synthesized have lost their affinity for the mitochondrial ribosome, while retaining their activity 

for the bacterial ribosome. These results testify to the feasibility of a combined genetic 

engineering/chemical synthesis approach to successfully alter drug-target interaction towards 

increased selectivity of aminoglycoside compounds.



WITOLD FILIPOWICZ 

Mechanism of the HuR-mediated reversal of miRNA repression in human cells 

Ellen I. Closs 1, Witold Filipowicz 2, Pradipta Kundu 2, Regula Dueggeli 2, 

Caroline Artus-Revel 2, Suvendra Bhattacharyya 2 

1 Department of Pharmacology, Johannes Gutenberg University, Germany 

2 Friedrich Miescher Institute for Biomedical Research, Switzerland 

MicroRNAs (miRNAs) are 21- to 24-nt-long non-coding regulatory RNAs expressed in 

metazoan animals and plants. In animals, miRNAs control gene expression 

post-transcriptionally by regulating translation or stability of mRNA in the cytoplasm. 

MiRNA-mediated translational repression is a reversible process in mammalian cells. As shown 

previously by us, target mRNAs with AU-rich regulatory elements (AREs) in the 3’UTR can be 

relieved from miRNA repression in response to different forms of cellular stress applied to 

human hepatoma Huh7 or HeLa cells. The ELAV family protein HuR, an ARE-binding protein, 

translocates from the nucleus to the cytoplasm in response to stress and its binding to target 

mRNA is essential for the relief of miRNA-mediated inhibition (Bhattacharyya et al., Cell 125, 

111-1124, 2006). 

We are investigating the mechanism of HuR-mediated effect on miRNA repression in human 

cells. Using mutants of HuR accumulating in the cytoplasm in the absence of stress and tumor 

cell lines constitutively accumulating endogenous HuR in the cytoplasm we were able to 

uncouple the HuR effect on miRNA repression from stress. We also found that Ago2 and HuR 

do not interact with each other and that their binding to target mRNA appears to me mutually 

exclusive. We are using recombinant wild-type and mutant HuR proteins and in vitro RISC 

assay to find out whether HuR has an effect on binding and activity of mi-RISC. Current status 

of these experiments will be discussed. 

22

ELISA IZAURRALDE 

23 


A C-terminal silencing domain in GW182 family proteins is essential for miRNA 

function in animal cells 

Elisa Izaurralde, Ana Eulalio, Felix Tritschler, Vincent Truffault, Isabelle Tournier, Daniela 

Lazzaretti, Latifa Zekri 

Max Planck Institute for Developmental Biology, Germany 

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; 

they interact with Argonaute proteins (AGOs) and are required for both the translational 

repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the 

GW182–AGO interaction in silencing, we generated protein mutants that do not interact and 

tested them in complementation assays. We show silencing of miRNA targets requires the 

N-terminal domain of GW182, which interacts with AGOs through multiple glycine-tryptophan 

(GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue 

silencing in D. melanogaster cells depleted of endogenous GW182. Conversely, silencing is 

impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with 

miRNAs. We further show that a GW182 mutant that does not localize to P-bodies, but 

interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells 

AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal 

AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as bipartite 

silencing region) are essential for silencing. Similarly, the three human GW182 paralogs (termed 

TNRC6A, TNRC6B and TNRC6C) interact with AGOs via their N-terminal domains and 

promote translational repression and/or degradation of miRNA targets through their C-terminal 

silencing domains. Together our results indicate that miRNA silencing in animal cells is 

mediated by AGOs in complex with GW182, and that P-body localization is not required for 

silencing.



THOMAS PREISS 

Identifying miRNA targets through changes in mRNA poly(A) tail length 

Thomas Preiss, Traude Beilharz, Jennifer Clancy, David Humphreys 

VCCRI, Australia 

It has been observed that miRNAs can trigger target mRNA deadenylation in fruit fly, zebrafish 

and mammalian systems. These studies further indicate that accumulation of an 

oligoadenylated intermediate is a widespread feature of the microRNA mechanism, whether 

the miRNA is markedly destabilizing the target or not. We, and others are pursuing implications 

of these findings for the miRNA mechanism. A further challenge in the field remains to develop 

facile experimental techniques to identify miRNA targets. Here we explore the potential of 

transcriptome-wide measurements of mRNA polyadenylation state as the basis for such 

screens. Using either northern blotting or the ligation-mediated poly(A) test (LM-PAT), we 

showed in HeLa cells or translation extracts that administration of an anti-miR against let-7 

causes a shift of a let-7 targeted reporter as well as endogenous HMGA2 mRNA from short 

(~20 adenosines) to markedly longer poly(A) tails. Based on these observations we posit that 

targets for a given miRNA can be identified by virtue of their change from a short-tailed state 

when the miRNA is active, to a long-tailed state when it is inactive. To measure 

transcriptome-wide changes in mRNA polyadenylation state between cellular conditions we 

have optimised conditions for tail length-based separation of mammalian mRNA on poly(U) 

sepharose beads, and are establishing procedures to analyse the resulting fractions by 

RNA-seq using SOLiD 3 technology. We will apply this methodology to situations where 

specific miRNAs are inhibited using anti-miRs, as well as to disruptions of the 

miRNA-processing pathway. Our approach may have several advantages over transcriptome 

analyses simply measuring changes in mRNA abundance. It is more likely to enrich for primary 

miRNA targets and it should also detect targets that are (near) exclusively repressed at the 

translational level. 

24

INCHEOL RYU 

25 


Eukaryotic Translation Initiation Factor 4G Mediates MicroRNA-Regulated 

Translational Gene Silencing 

Incheol Ryu, Ji Hoon Park, Oh Sung Kwon, Ki Young Paek, Sung Key Jang 

POSTECH, Korea 

MicroRNAs (miRNAs) are 21~23-nucleotide small noncoding RNAs and mediate 

post-transcriptional gene silencing by binding to their target mRNAs according to the 

sequence complementarity and recruiting miRNP complex to the target mRNAs. However, the 

molecular basis of the silencing is unclear. Here, we show that Ago2 interacts with eukaryotic 

translation initiation factors including eIF3, eIF4A, eIF4E, eIF4G and PABP. Among them, 

knockdown of eIF4G abrogates miRNA-mediated translational gene silencing and decreases 

the binding of Ago2 to the cap structure. The N-terminal and the middle domains of eIF4GI 

were responsible for the interaction with Ago2. Moreover, overexpression of eIF4GI 

dramatically increases the cap-binding affinity of Ago2. Taken together, the data suggest that 

eIF4G mediates miRNA-mediated gene silencing by promoting interaction of Ago2 with the cap 

structure.



ANIA WILCZYNSKA 

Investigating the microRNA pathway in Xenopus oocytes 

Ania Wilczynska1, Nancy Standart, Javier Armisen, Eric Miska 

Univerisity of Cambridge, United Kingdom 

Translational control mediated by specific RNA-binding proteins such as CPEB is critical in 

early development. Here we examine the possible role of the microRNA pathway in gene 

expression control in Xenopus oocytes. 

Firstly, reporter mRNAs injected into the cytoplasm are repressed ~ two-fold when tethered to 

the C-terminal domain of human GW1821; the cap- and poly(A)-dependence of this effect is 

being investigated. Secondly, deep sequencing of X. tropicalis small RNAs throughout 

oogenesis, verified in both X. tropicalis and laevis by Northern analysis, identified several 

moderately abundant and fully processed microRNAs (in addition to many piRNAs and 

endogenous siRNAs2,3), including miR-202-5p, highly enriched in ovary and testis in frog and 

mouse4,5. Injection of 202-5p AS LNA oligos, but not control oligos, accelerates meiotic 

maturation, which is delayed by injection of 202-5p mimic duplexes. Furthermore, the MI-MII 

transition is impaired by AS 202-5p LNA, but not control oligos. Our observations support the 

existence of an active microRNA pathway in oocytes. We are testing potential targets of 

202-5p, and other maternal microRNAs which may be involved in the resumption and 

completion of meiosis. 

1. Zipprich J, et al. 2009. Importance of the C-terminal domain of the human GW182 protein 

TNRC6C for translational repression. RNA 15, 781 

2. Armisen J, et al. Abundant and dynamically expressed miRNAs, endo-siRNAs and piRNAs 

in the African clawed frog Xenopus tropicalis; submitted 

3. Wilczynska A, et al. 2009. Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus 

female germline. RNA 15, 337 

4. Ro S, et al. 2007. Cloning and expression profiling of small RNAs expressed in the mouse 

ovary. RNA, 13, 2366 

5. Michalak P and Malone JH. 2008. Testis-derived microRNA profiles of African clawed frogs 

(Xenopus) and their sterile hybrids. Genomics. 91, 158 

26

ANNE-CATHERINE PRATS 

27 


FGF1 induction in myogenesis depends on novel cross-talks between IRES, 

promoter and 3’UTR 

Frederic Lopez 1, Anne-Catherine Prats 2, Nadera Ainaoui 2, Caroline Conte 2, Aurelie 

Delluc-Clavieres 2, Marie Khoury 2, Rania Azar 2, Yvan Martineau 2, Stephane Pyronnet 2 

1 IFR150, France 

2 Inserm U858, France 

Fibroblast growth factor 1 (FGF1) is involved in muscle development and regeneration, and 

required for muscle fiber formation. The FGF1 gene structure is complex as it contains four 

tissue-specific promoters allowing, by a process of alternative splicing, synthesis of four 

transcripts with distinct leader regions. Two of them contain internal ribosome entry sites 

(IRESs), which are RNA elements allowing mRNA translation to occur in conditions of blockade 

of the classical cap-dependent mechanism. 

We have investigated the molecular mechanisms regulating FGF1 expression during myoblast 

differentiation. We show that FGF1 is induced in differentiating myoblasts and regenerating 

mouse muscle, and that such induction is both transcriptional and translational, involving 

specific and simultaneous activation of FGF1 promoter A and IRES A at day 2 of differentiation, 

when cap-dependent translation is down-regulated. Furthermore, IRES activation is drastically 

increased by the presence of a mRNA 3’UTR element. 

Strikingly, we show that transcriptional and translational levels of FGF1 induction are 

molecularly coupled, as IRES-driven translation is clearly activated by a promoter cis-acting 

element. The mechanisms controlling these cross-talks of FGF1 IRES with promoter and 

3’UTR have been addressed by the biacore leading edge technology coupled with mass 

spectrometry, in order to identify protein complexes bound to FGF1 promoter DNA, as well as 

to FGF1 IRES and 3’UTR RNA during myoblast differentiation. Biacore technology turned out 

to be successful for protein recovery and identification. New data about identification of 

potential ITAFs regulating the FGF1 IRES activity will be presented. 

These resultas reveal a novel mechanism of regulation of IRES-dependent translation, involving 

both promoter and 3’UTR, which has dramatic consequences on a physiological event, that is, 

muscle development.



WILLIAM MERRICK 

Possible mechanism of regulation of IRES-mediated expression by eIF2A 

William Merrick, Yu Cao, Lucas Reineke, Diane Baus 

Case Western Reserve University, United States of America 

We have previously shown that eIF2A has the capability of repressing expression from the 

URE2 IRES in vivo by use of a knock out strain of yeast.1 Current studies have been 

addressing how this down regulation might be achieved. There are now five pieces of evidence 

that allow for the postulation of a model for this regulation: 1. the eIF2A mRNA disappears 

within 2-3 minutes with essentially any stress 2. eIF2A protein may or may not show enhanced 

degradation depending on the stress 3. eEF1A binds to the URE2 IRES element 4. by pull 

down experiments, eEF1A and eIF2A reciprocally bind each other (although each also pulls 

down several other proteins) 5. it appears that the interaction between eEF1A and eIF2A is 

through the C-terminal regions of each protein In the model to be presented, it is anticipated 

that the binding of eEF1A to the URE2 IRES-element facilitates the binding of eIF2A (and 

Met-tRNA) and that the resulting complex at the level of the 80S ribosome is slow to convert to 

an elongating ribosome With the down-regulation of eIF2A protein and/or activity, eIF2 is 

presumed to direct Met-tRNA binding in a manner that is much more efficient allowing for a 

10-fold increase in expression from this IRES element. Finally, we have preliminary evidence 

that the regulation of IRES-mediated expression is not exclusive to the URE2 IRES element but 

that eIF2A down-regulates expression from several other IRES elements as well. However, for 

most of the IRES elements with poly(A) stretches 5’ of the initiating AUG (identified by Gilbert et 

al.2), there appears to be no regulation by eIF2A. 1. Komar, A. A., Gross, S. R., Barth-Baus, 

D., Strachan, R., Hensold, J. O., Kinzy, T. G. and Merrick, W. C., J. Biol. Chem. 280, 

15601-15611, 2005. 2. Gilbert, W. V., Zhou, K., Butler, T. K. and Doudna, J. A., Science 317, 

1224-1227, 2007. 

28

CHRISTINE HOLT 

29 


Sub-cellular profiling reveals distinct and dynamic repertoire of growth cone 

mRNAs 

KH Zivrai, YCL Tung, M Piper, J Fawcett, GSH Yeo, Christine Holt 

Dept. of Physiology, Development and Neuroscience, University of Cambridge, United 

Kingdom 

Growing axons contain multiple mRNAs and cue-induced local translation in Xenopus retinal 

growth cones plays a key role in directed migration. The growth cone, at the leading tip of the 

axon, is a functionally distinct sub-cellular compartment that steers axon growth yet little is 

known about the repertoire of mRNAs and their developmental regulation. Here, we have used 

laser microdissection to capture the growth cones of embryonic retinal ganglion cells (RGCs) in 

two vertebrate species, Xenopus and mouse, and have performed unbiased genome-wide 

microarray profiling. We identify a surprisingly large number of growth cone mRNAs, Xenopus 

(n=958) and mouse (n=2187), representing 4.8-6% of the total number of transcripts on the 

arrays, and encoding proteins of diverse functions. Crucially, there is an overlap of 238 mRNAs 

present in both the Xenopus and mouse growth cones indicating a fair degree of conservation. 

Comparative profiling of mRNAs from laser-captured axon shafts versus growth cones 

revealed about 50 transcripts that are enriched specifically in the growth cone. The data were 

validated by both quantitative PCR and fluorescent in situ hybridization. Since retinal growth 

cones alter their responsiveness to guidance cues as they advance through the visual pathway 

through an age-dependent mechanism, we asked whether the mRNA pool changes 

dynamically with age. Comparative profiling of laser-captured ‘young’ (stage 24) versus ‘old’ 

(stage 32) Xenopus growth cones (approximately 12-18 hours difference) revealed 

unexpectedly large differences in the size and composition of mRNAs at the two ages. Thus, 

our studies reveal a dynamic repertoire of growth cone mRNAs that may contribute to steering, 

target selection and synapse formation in this specialized pre-synaptic compartment.



RAÚL MÉNDEZ 

CPEB1 regulates the expression of CPEB4 to complete meiosis 

Ana Igea, Raúl Méndez 

1 Center for Genomic Regulation (CRG), Spain 

The Maternal mRNAs that drive meiotic progression in oocytes contain short poly(A) tails and it 

is only when these tails are elongated that translation takes place. Cytoplasmic polyadenylation 

requires two elements in the 3’-UTR, the hexanucleotide AAUAAA and the cytoplasmic 

polyadenylation element (CPE), which recruits the CPE-Binding protein (CPEB1). However, not 

all CPE-containing mRNAs are activated at the same time during cell cycle, and 

polyadenylation is temporally and spatially regulated during meiosis. We have recently 

deciphered a combinatorial code that can be used to qualitatively and quantitatively predict the 

translational behavior of CPE-containing mRNAs (Piqué et al. 2008). This code defines positive 

and negative feed-back loops that generate waves of polyadenylation and deadenylation, 

creating a circuit of mRNA specific translational regulation that drives meiotic progression 

(Belloc and Méndez 2008). Using this code we have identified the mRNA coding for CPEB4, a 

member of the CPEB-familly of RNA-binding proteins, as a maternal mRNA regulated by 

cytoplasmic changes of its poly(A) tail length. We show that CPEB4 mRNA is a maternal 

mRNA transiently activated during metaphase I generating a pulse of CPEB4 synthesis that is 

required for the transition between the first and the second meiotic divisions. A model of 

sequential CPEB1 and CPEB4 activities in meiotic progression, where CPEB4 replaces CPEB1 

for the second meiotic division will be presented. REFERENCES: Piqué M, López JM, Foissac 

S, Guigó R, Méndez R. “A combinatorial code for CPE-mediated translational control”. Cell 

132:434-448 (2008). Eulàlia Belloc and Raúl Méndez. “A deadenylation negative feedback 

mechanism governs meiotic metaphase arrest”. Nature, 452(7190):1017-21 (2008) 

30

HOWARD LIPSHITZ 

31 


Control of mRNA translation and stability during early Drosophila development 


Drosophila Smaug (SMG) and Pumilio (PUM) are posttranscriptional regulators that regulate 

mRNA translation and/or stability. We previously reported that elimination of the majority of 

unstable maternal mRNAs in the early embryo requires SMG. RNP-IP microarray (‘RIP-chip’) 

experiments have now identified several hundred mRNAs that are associated with 

SMG-containing mRNPs, including a significant subset of the aforementioned stabilized 

mRNAs, consistent with a direct role for SMG in binding to, and elimination of, these 

transcripts. Similar methods are being used to assess PUM’s role in regulation of transcript 

stability and to identify direct targets. In addition, mass spectrometry of purified 

PUM-containing complexes, will identify proteins that participate in PUM-mediated 

posttranscriptional regulation. 

When Drosophila primordial germ cells (PGCs) bud, some germ plasm is incorporated into the 

underlying somatic cells but it is unclear how these cells avoid taking on germ-cell 

characteristics. SMG is enriched in novel mRNPs (‘S bodies’) apical to the nuclei of these 

posterior somatic cells. Live imaging of Vasa (VAS-GFP) simultaneous with Cherry-SMG, has 

shown that VAS is initially present in S bodies but then disappears. In contrast, several other 

germ-plasm components are stably maintained in S bodies, including nos mRNA and Oskar, 

Tudor and Germ cell-less proteins. Translational repression of nos mRNA by SMG in the S 

bodies is required to prevent posterior somatic cells from adopting germ-cell characteristics. 

Expression of NOS in these cells causes delamination from the posterior midgut epithelium, 

extrusion of lamellipodia, and co-migration with the PGCs. Thus S bodies function as sites of 

translational repression of the left-over germ plasm to distinguish somatic from germ-cell fates.



MARIA BARNA 

Translational specificity of ribosomal proteins regulates vertebrate embryonic 

development 

Toshihiko Shiroishi 1, Maria Barna 2, Nadya Kondrashov 2, Aya Pusic 2 

1 National Institute of Genetics, Japan 

2 University of California, San Francisco, United States of America 

Loss of function of distinct ribosomal proteins result in surprisingly specific phenotypic 

consequences and this effect is evolutionary conserved in Plants, Drosophila, Zebrafish, 

Mouse and Humans. How these phenotypes arise remains poorly understood and has not 

been directly linked to changes in gene expression at the level of translational control. Here we 

show that in three mouse mutants Tail-short (Ts), Tail-short shionogi (Tss) and Rabo torcido 

(Rbt) the Rpl38 gene, which encodes a small 8.1kD component of the 60S large ribosome 

subunit is found mutated. All three mouse mutants exhibit similar and dramatic 

tissue-patterning defects, including axial skeletal homeotic transformations. By establishing an 

unbiased polysome profiling approach, optimized for small embryos, we show that Rpl38 

selectively promotes the translation of a subset of homeobox (Hox) genes required to establish 

the mammalian body plan. We further show that the ability of Rpl38 to regulate gene 

expression is dictated by unique signatures present in the 3’Untranslated Region of target 

mRNAs. The tissue specific phenotypes and changes in Hox gene expression observed in Ts 

mutant embryos can be explained, at least in part, by enriched expression of Rpl38 in affected 

tissues such as somites, the precursors of vertebrae. Further quantitative analysis of all 79 

ribosomal proteins associated with both large and small ribosomal subunits reveals an 

unexpectedly dynamic and specific expression pattern during organogenesis. Collectively, 

these findings support the existence of a “ribosomal protein code” in which translational 

specificity of ribosomal proteins that show distinct patterns of expression may provide a new 

level of regulation in gene expression. 

32

MICHAEL SHEETS 

33 


Spatially regulated translation of the xCR1 mRNA in xenopus embryos by poly 

(A) independent mechanisms 

Amy Cook 1, Marvin Wickens 1, Michael Sheets 2, Yan Zhang 2, Kara Forinash 2, Jered 

McGivern 2, Brian Fritz 2 

1 University of Wisconsin Department of Biochemistry, United States of America 

2 University of Wisconsin Department of Biomolecular Chemistry, United States of America 

In vertebrate embryos the cripto protein xCR1 is a maternal cell-surface receptor required for 

nodal signaling. In Xenopus embryos the xCR1 protein is synthesized specifically by the animal 

cells and this differential accumulation of the xCR1 protein is due to vegetal cell-specific 

repression of xCR1 mRNA translation (Zhang et al MCB 2009). Surprisingly differential 

polyadenylation is not the primary determinant of this regulated translation. Vegetal cell-specific 

translational repression is mediated by a region of the xCR1 mRNA 3’UTR that contains both 

pumilio binding elements (PBEs) and binding elements for CUG-BP1. Mutation of either the 

PBEs or the CUG-BP1 sites in the xCR1 3’UTR abolishes binding by each respective protein 

and also eliminates translational repression of an mRNA reporter within vegetal cells. 

Immunoprecipitation RT-PCR experiments reveal that both pumilio and CUG-BP1 bind the 

xCR1 mRNA in embryos. Pumilio proteins often function in deadenylase complexes to 

negatively regulate mRNAs either by inhibiting translation or directing mRNA degradation. Our 

results suggest that pumilio and CUG-BP1 function together in a unique deadenylase complex 

that forms on the xCR1 mRNA 3’UTR. This complex is specialized for repressing the 

translation of maternal mRNAs in vegetal cells of Xenopus embryos independent of poly(A). In 

addition, we have found that translation of the maternal mRNA encoding another receptor for 

nodal signaling (the activin type-II-A receptor) is regulated similarly to the xCR1 mRNA. Thus 

the mechanisms that regulate xCR1 mRNA translation may also spatially regulate the 

translation of other maternal mRNAs encoding proteins important for nodal signaling. Our 

findings represent the first example of spatially regulated translation in controlling the 

asymmetric distribution of a maternal determinant in vertebrate embryos.



SEBASTIAN BAUMANN 

Microtubule-dependent mRNA transport during pathogenic development in 

Ustilago maydis 

Sebastian Baumann 1, Koepke Janine 1, Thomas Pohlmann 1, Kathi Zarnack 1, Michael 

Feldbrügge 1, Julian König 2 

1 Max Planck Institute for Terrestrial Microbiology, Germany 

2 MRC Laboratory of Molecular Biology, United Kingdom 

In the plant pathogen Ustilago maydis the formation of polar-growing filaments is essential for 

infection. This filamentation depends on the RNA-binding protein Rrm4 that assembles into 

particles shuttling bi-directionally along cytoplasmic microtubules. Particle formation and active 

transport by molecular motors is important for the function of the protein. Combination of in 

vivo UV crosslinking (CLIP) and λN-Gfp RNA live imaging revealed that Rrm4 mediates 

microtubule-dependent transport of distinct sets of transcripts encoding, e.g., the ubiquitin 

fusion protein Ubi1 or the small G protein Rho3. These mRNAs accumulate in directionally 

moving messenger ribonucleoprotein particles (mRNPs) that co-localise with Rrm4. 

Importantly, the CA-rich 3’ UTR of ubi1 identified by CLIP functions as zipcode during mRNA 

transport, as confirmed by λN-Gfp RNA live imaging. Furthermore, motile mRNPs are not 

formed when the RNA-binding domain of Rrm4 is deleted, although the protein is still shuttling. 

Thus, Rrm4 constitutes an integral component of the transport machinery. Loss of mRNA 

transport correlates with defects in polar growth indicating that Rrm4-mediated mRNP 

trafficking is required for polarity. This is the first example for long-distance mRNA transport in 

fungi and our data suggest that the fundamental principles are conserved among fungi, plants 

and animals. 

34

PETER LUKAVSKY 

35 


A’-form RNA helices drive microtubule-based mRNA transport in Drosophila 

Inbal Ringel 1, David Ish-Horowicz 1, Peter Lukavsky 2, Simon Bullock 2 

1 Cancer Research, United Kingdom 

2 MRC LMB, United Kingdom 

Microtubule-based mRNA transport is widely used to restrict protein expression to selected 

regions in the cell, and has important roles in defining cell polarity, axis determination and for 

neuronal function. However, the molecular basis of recognition of cis-acting mRNA localization 

signals by motor complexes is poorly understood. We have used NMR spectroscopy to 

describe the structure of the 44 nt RNA element responsible for dynein-mediated localization of 

Drosophila fs(1)K10 (K10) transcripts. The K10 signal adopts a stem-loop with unusual 

structural features. Stacking interactions of purine bases within canonical, double-stranded 

RNA helices give rise to base pair inclinations and widened major grooves consistent with 

A’-form conformation. We investigate the nature and functional importance of these features 

by structure determination of four mutant K10 elements and by monitoring in vivo the transport 

of a large series of mutant RNAs. Our data suggest that two spatially registered widened 

major grooves represent the binding sites for the transport machinery, the occupancy of which 

determines the RNA distribution within the cell. This study also demonstrates that 

double-stranded RNA with regular base pairs has unappreciated structural complexity capable 

of mediating selective recognition and thereby assigns a key biological function to the A’-form 

RNA conformation.



GRECO HERNANDEZ 

Mextli is a novel eIF4E-binding protein from Drosophila 

Greco Hernandez, Mathieu Miron, Gritta Tettweiler, Nahum Sonenberg, Paul Lasko 


eIF4E is a key regulator of translation. eIF4E-binding proteins (4E-BPs) play a critical role in the 

regulation of eIF4E. They bind the dorsal convex surface of eIF4E to form a translationally 

inactive eIF4E/4E-BP complex. Upon stimulation of cells with hormones or growth factors, 

4E-BPs become phosphorylated and dissociate from eIF4E, rendering eIF4E available to 

interact with eIF4G to form the translationally active eIF4G/eIF4E complex. Recently, the 

interaction of eIF4E with other proteins, including 4E-transporter (4E-T) and maskin, has been 

shown to regulate specific cellular or developmental processes. For these reasons, we are 

searching for additional eIF4E binding proteins involved in Drosophila development. From a 

far-western genomic screen on a embryonic cDNA library, using 32P-labeled 

FLAG-HMK-eIF4E1 as a probe, we identified a novel eIF4E-binding protein with no similarity to 

other eIF4E-binding proteins such as eIF4G, 4E-T and the 4E-BPs. It possesses a canonical 

eIF4E binding site YXXXXLL that, when mutated to AXXXXAA, the interaction with different 

Drosophila eIF4Es is abrogated. The novel eIF4E binding protein has a predicted molecular 

weight of 70.1 kDa and is encoded by three alternatively-spliced mRNAs encoded by a single 

gene. The functional features of this protein in the ovary and embryos will be presented 

36

ANA VILLLALBA 

A novel, non-canonical mechanism of cytoplasmic polyadenylation in 

Drosophila 

Olga Coll, Fátima Gebauer, Ana Villlalba 

Center for Genomic Regulation, Spain 

37 


Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. This 

process has been thoroughly studied during vertebrate oocyte maturation, where two 

sequences in the 3’UTR are required: the conserved AAUAAAA hexanucleotide and the U-rich 

cytoplasmic polyadenylation element (CPE). Cytoplasmic polyadenylation also occurs during 

early embryogenesis, but the sequences and factors that function at this time of development 

are not well understood. Using an in vitro system derived from Drosophila embryos, we show 

that the vertebrate cytoplasmic polyadenylation signals function in Drosophila. Surprisingly, 

deletion of these sequences in a known Drosophila substrate, toll mRNA, did not affect 

polyadenylation. Extensive mutational analysis showed that a proximal region of toll 3’ UTR, 

referred to as PR, is critical for polyadenylation. Competition experiments indicated that PR 

interferes with cytoplasmic polyadenylation of toll in vitro and in vivo, leading to reduced viability 

of early embryos. Importantly, PR did not affect polyadenylation of a typical CPE- and 

hexanucleotide- dependent substrate. These data indicate that toll mRNA is polyadenylated by 

a non-canonical mechanism, and suggest that a novel machinery functions for cytoplasmic 

polyadenylation during Drosophila embryogenesis.



KOBI ROSENBLUM 

Translational Control in the Gustatory Cortex Determines the Stability of a 

Taste Memory 

Haifa University, Israel 

The off-line processing of acquired sensory information in the mammalian cortex is an example 

for the unique way biology creates to compute and store information which guides behavior. 

The relatively short temporal phase in the process (i.e. hours following acquisition) is defined 

biochemically by its sensitivity to protein synthesis inhibitors. Until recently this negative 

definition of molecular consolidation did not reveal the details of the endogenous processes 

taking place, minutes to hours, in the neurons and circuit underlying a given memory. We use 

taste learning paradigms in order to study this process of molecular consolidation in the 

gustatory cortex. 

Recent results, from our laboratory, obtained from genetic, pharmacological, biochemical, 

electrophysiological and behavioral studies demonstrate that translational control, at the 

initiation and elongation phases of translation, plays a key role in the process of molecular 

consolidation. Moreover, this spatially and temporally regulated translation control modifies 

both general and synaptic protein expression that is crucial for memory stabilization. We 

propose a model to explain the interplay between regulation of initiation and elongation phases 

of translation and demonstrate that in certain situations cognitive enhancement can be 

achieved. 

38

KENT DUNCAN 

The SXL-UNR Corepressor Complex Uses a Novel PABP-Mediated 

Mechanism to Inhibit Ribosome Recruitment to msl-2 mRNA 

Kent Duncan, Claudia Strein, Matthias W Hentze 

EMBL, Germany 

39 


Modulation of translation initiation by proteins bound to specific sequence elements in mRNA 

3' untranslated regions (UTRs) is a common mode for control of eukaryotic gene expression. 

Nevertheless, molecular mechanisms by which 3'UTR regulators influence initiation events on 

the mRNA 5' end remain largely unclear. Regulation of Drosophila MSL-2 protein synthesis 

provides a powerful model system to address this key issue in translational control. Inhibition of 

MSL-2 synthesis is crucial for female viability and requires interaction of the female-specific 

RNA binding protein sex-lethal (SXL) with specific sites in the msl-2 mRNA 3'UTR. SXL recruits 

the protein UNR to adjacent sites, and this 3'UTR repressor complex inhibits recruitment of the 

small ribosomal subunit to the 5' end of the mRNA by an unknown mechanism. We have 

investigated this mechanism using functional assays in Drosophila embryo cell-free translation 

extracts. Our results reveal a key role for the poly(A) binding protein (PABP), a translational 

activator, in this inhibitory mechanism. Efficient msl-2 mRNA silencing via the 3'UTR requires 

both a poly(A) tail and PABP function, and we find that UNR directly interacts with PABP. To 

investigate how the SXL-UNR repressor complex and PABP affect RNP composition during 

early steps in translation initiation, we established two general assays based on the GRNA 

affinity chromatography approach: (1) a direct biochemical assay for synergistic recruitment of 

eIF4F by the cap and poly(A) tail and (2) a novel method to monitor specific mRNA association 

of both the small and large ribosomal subunits via quantitative real-time RT-PCR analysis. We 

find that the repressor complex targets small subunit recruitment after poly(A)-mediated 

recruitment of eIF4E/G. Our results uncover an important regulatory mechanism of Drosophila 

dosage compensation and highlight a new mode of PABP-dependent translational control by 

3'UTR-bound regulatory proteins.



FATIMA GEBAUER 

Regulatory networks controlled by Drosophila UNR 

Marija Mihailovich 1, Irina Abaza 1, Federico Zambelli 2, Giulio Pavesi 2, Fatima Gebauer 1 

1 Center for Genomic Regulation, Spain 

2 Department of Biomolecular Science and Biotechnology, University of Milano, Italy 

UNR is a conserved, cytoplasmic protein containing five cold shock domains that serve to bind 

single stranded nucleic acids. In Drosophila, UNR has been identified as a major regulator of 

X-chromosome dosage compensation, the process by which expression of X-linked genes is 

equalized in males and females. In this context, UNR performs sex-specific opposite functions: 

in female flies, UNR inhibits the translation of msl-2 mRNA, which encodes a component of the 

dosage compensation complex (DCC), and hence blocks dosage compensation. In males, 

UNR promotes DCC assembly on the X chromosome by a mechanism that is independent of 

msl-2 translational regulation. 

To gain insight into the sex-specific roles of UNR, we set to identify UNR regulatory networks. 

We performed high scale immunoprecipitation of UNR from either male or female adult 

extracts, and identified UNR-bound RNAs by microarray analysis and Solexa deep sequencing. 

Curiously, UNR binds to non-overlapping set of transcripts in males and females, suggesting 

that UNR largely contributes to sex-specific gene expression. Proteins encoded by UNR 

mRNA targets fall into a variety of functional groups, including important transcriptional and 

post-transcriptional regulators. Interestingly, mRNAs for four other factors involved in dosage 

compensation (mle, su(var)3-7, msl-1 and Mtor) were selected specifically in females. 

Knock-down assays in female Kc cells confirmed that UNR regulates the expression of at least 

some of these transcripts. Our data suggest that UNR regulates a post-transcriptional operon 

to repress dosage compensation in female flies. 

40

SHELTON BRADRICK 

41 


Identification of gemin5 as a novel cap-binding protein that associates with 

coding and non-coding RNAs 

Shelton Bradrick, Matthias Gromeier 


RNA molecules synthesized by RNA polymerase II bear a 7-methylguanosine (m7G) cap 

structure added co-transcriptionally to the 5’ end. Through association with trans-acting 

effector proteins, the m7G cap participates in multiple aspects of RNA metabolism including 

localization, translation and decay. Although m7G mediates critical roles in gene regulation, it is 

presently unclear whether the full complement of cellular proteins capable of binding the cap 

structure has been defined. Utilizing an unbiased cap-affinity chromatography approach, we 

identified the non-canonical RNA-binding protein gemin5, a predominantly cytoplasmic 

component of the survival of motor neuron (SMN) complex, as a factor capable of direct and 

specific interaction with the m7G cap. Gemin5 bound m7G-linked resin in the absence of 

detectable eIF4E or the full SMN complex. Moreover, gemin5 specifically cross-linked to 

radiolabeled cap structure in a manner that depended upon integrity of WD40 motifs located 

within its N-terminal region. Molecular characterization of endogenous gemin5 RNP complexes 

by microarray and RT-PCR analyses unexpectedly revealed the presence of mRNAs in addition 

to the non-coding U1 snRNA. Further, cell-free translation assays demonstrated that gemin5 is 

capable of specifically inhibiting cap-dependent mRNA translation through its affinity for the 5’ 

cap. In summary, these findings identify a new m7G cap-binding protein and implicate gemin5 

as a post-transcriptional regulator that participates in diverse RNP complexes containing 

coding and non-coding RNAs.



MICHAL SHAPIRA 

Evolutionary diversity of the trypanosomatid cap4-binding complex – a 

potential drug target against trypanosomatids? 

Michal Shapira 1, Yael Yoffe 1, Alexandra Zinoviev 1, Melissa Leger 2, Gerhard Wagner 2, 

Joanna Zuberek 3, Edward Darzynkiewicz 3 

1 Ben Gurion University of the Negev, Israel 

2 Harvard Medical School, United States of America 

3 Warsaw University, Poland 

All trypanosomatids possess a highly modified cap-4 structure at the 5’ ends of their mRNAs, 

donated by the spliced leader RNA during trans-splicing. We have been analyzing the 

cap-binding complex of Leishmania. Subunits of the trypanosomatid cap binding complex 

have evolved to bind the unique cap-4 structure and are therefore structurally diverged from 

their higher eukaryote orthologues. We identified four eIF4E homologues in the TriTryp genome 

database and at least five eIF4G candidates. The evolutionary diversity of the trypanosomatid 

eIF4E proteins is emphasized by their inability to functionally complement the mutated yeast 

protein, unlike the Drosophila and Arabidopsis eIF4Es. In the absence of a functional system 

for in vitro translation in trypanosomatids, their functional annotation is complex and was based 

on monitoring the binding kinetics to a synthetic cap-4 analogue, migration on sucrose 

gradients and their ability to generate eIF4E-eIF4G complexes. LeishIF4E-1 and LeishIF4E-4 

are therefore most probably the basal translation initiation factors; LeishIF4E-1 seems to also 

function under stress conditions. The eIF4G homologue of Leishmania was identified by pull 

down assays (LeishIF4G-3) and it too reflects evolutionary changes. It contains a conserved 

MIF4G domain, but has a short N-terminus that is responsible for binding the parasite eIF4E, 

but not the human protein. In higher eukaryotes the eIF4E-eIF4G interaction is based on a 

conserved peptide signature [Y(X4)LΦ]. A parallel, but extensively variable eIF4E binding 

peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). In addition to Y20 and L25, a strict 

requirement for F23 was monitored, whereas the hydrophobic amino acid (Φ) is dispensable. 

The eIF4E-eIF4G interaction in Leishmania was also confirmed by NMR studies. In view of 

these diversities, the characterization of the parasite eIF4E-eIF4G interaction may serve as a 

novel target for inhibiting Leishmaniasis. 

42

GREGORY BOEL 

YjjK, a member of the ATP binding cassette superfamily is a novel 

transcriptional factor 

43 


Gregory Boel, Magali Cottevielle, Joachim Frank, John F. Hunt, Samuel K. Handelman, 

Paul C. Smith 1 


The soluble tandem ATP-Binding Cassette (stABC or ABC-F) family contains soluble 

cytoplasmic proteins that consist of two fused ABC domains. Unlike most ABC superfamily 

members, they are not involved in transport across membranes. The biochemical function of 

the proteins in this family is unknown, although they are most closely related to the 

fungal-specific elongation factor 3 (EF3). The X-ray crystal structure of YjjK, an ABC-F family 

member from Escherichia coli, shows a head-to-tail dimer. This molecular arrangement 

suggests that concerted binding of four ATP molecules triggers a large mechanical 

rearrangement in this complex. Furthermore, each ABC domain features an “Arm” that extends 

from the protein core. Investigation into the cellular function of YjjK shows that this protein is 

associated with the ribosome during translation. The crystal structure of YjjK was used to 

design biochemical experiments, which demonstrated that the protein is functionally 

associated with the ribosome. An engineered variant of YjjK, which strongly binds to the 70S 

ribosome, was used to purify the YjjK-70S ribosome complex. Cryo-EM image reconstruction 

studies of this complex show that a monomer of YjjK binds between the interface of the large 

and small ribosomal subunits blocking the binding site for the standard translational elongation 

factors. An E. coli strain inactivated for the yjjK gene shows different phenotypes including 

motility deficiencies and gain of growth for one carbon source. We are now using high 

throughput phenotypic profiling of a YjjK knockout strain to establish the physiological 

consequences of YjjK function. In addition, microarray profiling and whole-cell proteomics will 

be used to establish the underlying changes in mRNA transcript and protein levels which 

should be readily correlated with the observed phenotypic properties.



LYUBOV RYABOVA 

Function of RISP in virus-induced translation reinitiation 

Lyubov Ryabova, Odon Thiébeauld, Mikhail Schepetilnikov, Angèle Geldreich, Mario Keller 

Institut de Biologie Moléculaire des Plantes (IBMP), UPR CNRS 2357, France 

Eukaryotic ribosomes usually do not translate the downstream ORF of a polycistronic RNA, if 

the first ORF is long. The exceptional case was discovered in the Cauliflower mosaic virus, 

where transactivator/viroplasmin (TAV) transactivates translation of viral and artificial 

polycistronic mRNAs. TAV participates in multiple interactions with host factors: eIF3 via 

subunit g, the 60S ribosomal subunit and a plant protein -reinitiation-supporting protein (RISP). 

Interaction of TAV with eIF3 and ribosomal protein L24 is required for its function in 

transactivation, where TAV might prevent loss of eIF3 from the translating ribosome after the 

first initiation event and participate in recruitment of the ternary complex and 60S for the 

reinitiation event. RISP can interact with TAV via its central domain that was proven to be 

essential for transactivation. RISP may physically interact with eIF3 via subunits a/c, and with 

60S via ribosomal protein L24 in vitro and in vivo. TAV-containing polysomes are specifically 

enriched with RISP and eIF3 in infected plants. Overexpression of RISP enhances 

TAV-mediated transactivation activity in plant protoplasts. TAV mutant that can not bind RISP 

is not active in transactivation. The GST-pull down and IP assays were used to analyze the 

interaction network between TAV, RISP, eIF3, L24 or 40S. The RISP/eIF3 complex interacts 

with 40S; eIF3/RISP/L24 and eIF3/TAV/RISP/L24 complex formation can be demonstrated in 

vitro. We speculate that RISP acting together with TAV bridges interactions between eIF3 and 

60S to achieve the reinitiation event. We suggest that RISP can be considered as a novel 

component of the translation machinery. Recently, we discovered a link between TAV and 

protein kinase TOR, which is required for TAV-mediated transactivation and assists virus 

propagation. 

44

PHILIP FARABAUGH 

45 


Effects of haploinsufficiency of ribosomal protein and assembly factor genes 

on cellular physiology mediated by RACK1 

David Garfinkel 1, Arun Dakshinamurthy 1, Philip Farabaugh 2, Susmitha Suresh 2 

1 National Cancer Institute, United States of America 

2 University of Maryland Baltimore County, United States of America 

Haploinsufficiency for ribosomal proteins occurs in human genetic diseases including 

Diamond-blackfan anemia, Schwachman-Bodian-Diamond syndrome and 

Hoyerall-Hreidarsson syndrome. This haploinsufficiency causes developmental malformation, 

immunodeficiency, premature aging and susceptibility to cancers. Though the etiology of these 

symtoms is unknown although they appear to result from defects in ribosome biogenesis or 

function. 

Deletions of non-essential or duplicated genes encoding ribosomal proteins and ribosome 

assembly factors alter Ty1 retrotransposon transposition in the yeast Saccharomyces 

cerevisiae. Ty transposition requires programmed +1 translational frameshifting between the Ty 

gag and pol genes. Frameshifting is reduced or increased ~2 fold in many of the mutants but 

not universally, implying that it is not the proximal cause of the defects. 

A clue to the origin of the phenotype came from our finding that the few mutations causing 

increased transposition include one targeting the ribosomal protein RACK1 (ASC1 in yeast). 

RACK1 is a 40S ribosomal protein that is a receptor for a variety of protein kinases; it binds 

near the RNA helicase center at the opening of the mRNA entrance channel. In yeast, RACK1 

is also the Gβ protein that represses the activity of the Gpa2 Gα. Gpa2 activates the Cyr1 

adenyl cyclase and, through it, the Tpk2 isoform of protein kinase A. Cyr1 and Tpk2 are also 

activated by Ras2. Significantly, null mutations of Gpa2, Ras2 and Tpk2 all show reduced Ty 

transposition, opposite that of asc1Δ. 

We are testing if RACK1 monitors the state of ribosome biogenesis and signals through its 

interaction with Gpa2, Cyr1 and Tpk2 to regulate Ty transposition and perhaps other aspects 

of cell physiology. We believe that a similar signalling system may be the cause of the disease 

effects of ribosome protein deletions in humans.



VITALY POLUNOVSKY 

Translational Control of Malignancy in the Murine Models Epithelial 

Carcinogenesis: The Role of the Translational Repressors 4E-BPs 

Ola Larsson 1, Vitaly Polunovsky 2, Yong Kim 2, Linda Von Weymarn 2, Danhau Fan 2, Jon 

Underwood 2, Stiven Hecht 2, Svetlana Avdulov 2, Peter Bitterman 2 


2 University of Minnesota, United States of America 

Translational control plays a key role in cancer genesis and progression. Whereas the 

oncogenic function of the initiation complex eIF4F has been firmly established, the role of the 

eIF4F antagonist’s 4E-BPs remains disputable. To address this issue in the context of the 

whole mammalian organism, we developed two complementary models of 4E-BP1-regulated 

tumorigenesis by using mice genetically engineered to:(a) luck of 4E-BP1 and 4E-BP2 

(4ebp1-/-/4ebp2-/-); (b) express the hypophosphorylated version of 4E-BP1 (4E-BP1A37/A46) 

in mammary glands. (a) The 4E-BP deficient state per se leads to pro-oncogenic, 

genome-wide skewing of the molecular landscape - with translational activation of genes 

governing angiogenesis, growth and proliferation; and translational activation of the precise 

cytochrome p450 enzyme isoform (CYP2A5) that bioactivates the tobacco carcinogen NNK 

into mutagenic metabolites. When challenged with NNK, 4ebp1-/-/4ebp2-/- mice develop 

5-fold more tumors than their wild type counterparts. (b) Mice expressing 4E-BP1A37/A46 

under control of the mammary specific promoter were bred to a transgenic strain prone to 

breast cancer by the ErbB2 oncogene. Bitransgenic F1 animals developed tumors with kinetics 

similar to those of monotransgenic erbb2/neu mice. Strikingly, expression of the 

4E-BP1A37/A46 transgene was gradually down regulated in the course of ErbB2-induced 

tumors formation in a manner similar to silencing of known tumor suppressor genes during 

breast tumorigenesis. Taken together, these data provide in vivo proof that 4E-BPs operate at 

the translational control checkpoint in tumor defense. 

46

ROBERT RHOADS 

47 


Effects on translation initiation and malignant transformation of MMTV insertion 

into the eIF3e gene 

Robert Rhoads 1, David Chiluiza 2, Robert Callahan 3 

1 LSU Health Sciences Center, United States of America 

2 LSUHSC-S, Shreveport, LA, United States of America 

3 National Cancer Institute, Bethesda, MD, United States of America 

An “extrachromosomal influence” on mammary tumor development first observed in 1936 was 

later attributed to mouse mammary tumor virus (MMTV), but insight into the mechanism was 

not obtained until it was shown that MMTV integrates into a small number of genomic sites. 

One common integration site, Int-6, encodes the “e” subunit of eIF3. Our laboratory recently 

found that eIF3e forms part of the binding surface for eIF4G. We speculated that truncated 

eIF3e protein (eIF3e-sh) interferes with association of eIF3-eIF4G and the resulting 

dysregulation of cap-dependent translation is responsible for tumorigenesis. We found that 

HC11 expressing eIF3e-sh exhibited reduced protein synthesis, growth in soft agar, resistance 

to apoptosis, and formation of nodular growths in nude mice. With an exogenous bicistronic 

mRNA we showed that cap-dependent firefly luciferase was reduced compared to HCV 

IRES-dependent renilla luciferase. To more closely model MMTV integration, we inserted 

sequences encoding eIF3e-sh at a single-copy Cre recombinase site in mouse 3T3 cells. 

Expression of eIF3e-sh diminished protein synthesis and decreased cap-dependent versus 

HCV-IRES-dependent translation. Exogenously introduced mRNAs do not undergo a “nuclear 

experience” and may not behave like endogenous mRNAs, so we measured the polysome 

distribution of endogenous mRNAs. We found that GAPDH mRNA (cap-dependent) shifted to 

lower polysomes in eIF3e-sh-expressing cells, indicating a decrease in initiation rate relative to 

elongation/termination, while the cellular IRES-containing mRNAs for c-Myc and the X-linked 

inhibitor of apoptosis (XIAP) were not affected. We speculate that expression of eIF3e-sh tips 

the balance toward cap-independent translation, thereby causing upregulation of known 

oncoproteins such as c-Myc and inhibitors of apoptosis such as XIAP. (Supported by NIH 

Grant R01GM20818)



ROBERT SCHNEIDER 

AUF1 links the chronic inflammatory response to telomere maintenance, 

premature aging and tumorigenesis through control of short-lived mRNA 

stability 

Robert Schneider, Sadri Navid, Pont Adam 


Regulation of telomere length plays an essential role in preventing premature senescence and 

maintaining genetic stability. Several heterogeneous ribonucleoproteins have been implicated in 

telomere length regulation based on in vitro studies. Here we show that AUF1-deficient 

(knockout) mice exhibit a striking increase in premature ageing phenotypes, cellular 

senescence and tumorigenesis. Litters from late generation heterozygous crossings show a 

sharp decrease in the frequency of AUF1-/- offspring, which exhibit premature senescence 

and mortality in the first month of life. Late generation AUF1-/- mice demonstrate shorter 

telomere lengths, an increase in the frequency of chromosomes without detectable telomere 

signals and numerous chromosomal abnormalities. AUF1-/- embryonic fibroblasts (MEFs) 

exhibit increased senescence, DNA damage foci at telomeres and activation of the DNA 

damage response. Premature aging, increased tumorigenic properties of AUF1-/- MEFs and 

increased tumor incidence in AUF1-/- mice are associated with extensive genetic alterations, 

particularly the loss of p16ink4a and p21CIP mRNA destabilization, key cell cycle regulators. 

AUF1 is also shown to regulate telomerase activity through the abundance of its associated 

RNA. Backcrossing late generation AUF1-/- mice for one generation with wild type mice 

rescues these defects, indicating the central role for AUF1 in maintenance of normal aging and 

telomere stability. 

48

HARALD KÖNIG 

Interfering with translation of transcripts enhances their splicing 

Harald König, Nico Braunegger, Helga Olinger 

Forschungszentrum Karlsruhe GmbH, Germany 

49 


The cell nucleus and nucleus-cytosol compartmentalization are defining hallmarks of eukaryotic 

cells. Although their origins are unclear, they are thought to have evolved under the pressure to 

segregate intron-containing transcripts and emerging pre-mRNA splicing from translation by 

ribosomes. Whether such interference between translation and splicing has existed is, 

however, not known. If so, its remnants may be still detectable in present-day eukaryotic cells, 

and might give hints on how strong a driving force for cell evolution this interference could have 

been. We report here that interfering with translation of endogenous transcripts enhances their 

splicing in vivo. Transcript-specific inhibition experiments and in-vivo analysis of 

ribonucleoprotein complexes argue in favor of a cis-acting mechanism underlying this effect. 

Our results point to interfering forces on transcripts between translation and the pre-mRNA 

splicing machinery, that can apparently not be completely prevented by current eukaryotic cell 

architecture and function.



MICHAEL KIEBLER 

Functional Characterization of Neuronal RNA granules and their role in 

dendritic RNA localization 

Keiryn Bennett 1, Giulio Superti-Furga 1, Michael Kiebler 2, Daniela Karra 3, Georgia Vendra 3, 

Martin Mikl 3 

1 CeMM, OEAW, Austria 

2 Medical University of Vienna, Austria 

3 MUW, Austria 

In mature hippocampal neurons, a small subset of RNAs localizes to dendrites in the form of 

ribonucleoprotein particles (RNPs). Using the RNA-binding proteins Staufen2 and Barentsz 

(MLN51) as neuronal RNA granule markers, we isolated RNPs from rat brain via affinity 

purification. This allowed us to identify – besides factors that have been implicated in RNA 

localization – protein components with known roles in mRNA stability, mRNA decay, 

translational control and small RNA-mediated translational silencing and RNA degradation. 

Using known P-body markers, e.g. DDX6/p54, Rck or Ago2, we have started to characterize 

their relationships to the neuronal RNA granules involved in RNA transport. In parallel, we are 

currently analyzing the RNA composition of these Stau2 and Btz granules in detail. In order to 

address whether different dendritic transcripts reside in the same or distinct neuronal RNPs, 

we are performing double in situ hybridization and microinjection of fluorescently labeled RNA 

in two colors, in primary neurons in culture. This allowed us to assess RNA sorting in individual 

dendritic RNPs. Our data supports the hypothesis that individual transcripts localize as 

oligomers, rather than large complexes, in the dendrites of mammalian neurons. 

50

ELENA CONTI 

Structural studies of nonsense mediated mRNA decay 

Max Planck Institute of Biochemistry, Germany 

51 


Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control process that subjects 

mRNAs with premature translation termination codons (PTCs) to rapid degradation. From a 

mechanistic standpoint, NMD factors are recruited to an mRNA when translating ribosomes 

recognize a stop codon as premature by sensing the presence of a second signal 

downstream. While the three central NMD effectors (the proteins UPF1, UPF2 and UPF3) are 

conserved across species, the nature of the downstream signal varies. The most common and 

best-studied signal that is required for efficient NMD in mammals is the exon junction complex 

(EJC). We have been addressing different steps of the NMD pathway by structural studies. The 

talk will focus on recent results, in particular on the mechanism by which the UPF3b 

component of the surveillance complex bridges the interaction to the EJC.



OLIVER MÜHLEMANN 

SMG6-mediated endonucleolytic cleavage of nonsense mRNA in human cells 

Søren Lykke-Andersen 1, Torben Heick Jensen 1, Oliver Mühlemann 2, Andrea Eberle 2, Lukas 

Stalder 2 

1 Aarhus University, Denmark 

2 University of Bern, Switzerland 

Messenger RNAs on which translation terminates aberrantly, at premature termination codons 

(PTCs) or at physiological termination codons too far away from the poly(A) tail, are recognized 

and rapidly degraded by a process called nonsense-mediated mRNA decay (NMD). Whereas a 

unified model for identification of NMD substrates emerged over the last few years, 

degradation mechanisms of NMD have been suggested to differ between species. In 

Drosophila, NMD is initiated by endonucleolysis near the PTC, whereas the current view posits 

that NMD in yeast and human cells occurs by exonucleolysis from one or both RNA termini. 

However, we found that degradation of human nonsense mRNAs can also be initiated by 

PTC-proximal endonucleolytic cleavage. In addition, we identified the metazoan-specific NMD 

factor SMG6 as the responsible endonuclease by demonstrating that mutation of conserved 

residues in its nuclease domain - the C-terminal PIN motif – abolishes endonucleolysis in vivo 

and in vitro. These data reveal a new degradation pathway for nonsense mRNA in human cells 

and suggest that endonucleolytic cleavage is a conserved feature in metazoan NMD. 

Furthermore, SMG6 does not reside in P-bodies and consistent with the evidence for 

decapping-independent SMG6-mediated endonucleolytic decay of human nonsense mRNAs, 

we showed that microscopically detectable P-bodies are not required for NMD in HeLa cells. 

52

FULVIA BONO 

Structural analysis of Mago-Y14 import receptor 

53 


Judith Ebert 1, Esben Lorentzen 1, Elena Conti 1, Marlene Gruenwald 2, Fulvia Bono 2, Cátia 

Igreja 2 

1 Max Planck Institute for Biochemistry, Germany 

2 Max Planck Institute for Developmental Biology, Germany 

The bulk of protein and RNA transport in and out of the nucleus is performed by karyopherins. 

These proteins are generally subdivided into importins and exportins depending on the 

direction of their transporting movement across the nuclear pores. The directionality is 

achieved through a RanGTP gradient. High RanGTP levels in the nucleus dissociate import 

cargo complexes and promote the binding of export cargos to exportins. Most karyopherins 

are unidirectional transporters, with the exception of Exportin 5 and Importin 13, which mediate 

bidirectional transport. Importin 13 imports several cargos into the nucleus, including the 

Mago-Y14 complex, a core component of the Exon Junction Complex (EJC), and it also 

exports the translation initiation factor eIF1A in a trimeric complex with RanGTP. Even though 

there is ample structural information on unidirectional karyopherins, an unsolved structural 

problem has been so far the mechanism of action of bidirectional transport receptors. We 

present the structure of Importin13 in complex with Mago-Y14, showing how the heterodimeric 

cargo is specifically recognized. We also present the structure of Importin13 in complex with 

RanGTP, which gives insight into how the dissociation of the Mago-Y14 cargo in the nucleus is 

achieved. In general, Importin 13 shows mixed features between importins and exportins in 

RanGTP and cargo binding.



GABRIELE NEU-YILIK 

Premature termination codons within exon 1 of the human beta-globin mRNA 

create short ORFs permissive for reinitiation 

Niels Gehring 1, Gabriele Neu-Yilik 2, Beate Amthor 2, Matthias W. Hentze 2, Andreas E. 

Kulozik 2 

1 EMBL, Germany 

2 MMPU EMBL/University of Heidelberg, Germany 

The analysis of nonsense mutated β-globin transcripts has revealed important functional 

aspects of NMD - such as the necessity of splicing and translation, the 50 nt rule, and the 

NMD resistance of last exon nonsense mutations - but also interesting examples of NMD 

insensitive transcripts that cannot be explained by current models of NMD. Specifically, 

experiments in transfected MEL and HeLa cells indicated that β-globin transcripts with 

nonsense mutations in the first of three exons are NMD insensitive (Romão et.al., Blood, 2000; 

Inácio et al., JBC, 2004). This observation challenged the general view that a splice event 3’ to 

the premature termination codon (PTC) suffices to trigger NMD. 

We have systematically analyzed a series of PTCs in exon 1 of the human β-globin gene and 

identify a sharp border between codons 23 and 26 in the first exon, 3’ to which PTC mutations 

trigger NMD whereas they do not if positioned 5’ to this border. Using venus-tagged β-globin 

genes, we show by immunoblotting and by flurorescence microscopy, that translation 

termination at PTCs within this border induces reinitiation at codon 55Met in exon 2. Mutation 

of this methionine codon both abrogates reinitiation and directs previously NMD-insensitive 

transcripts to NMD. Likewise, extensions or truncations of the ORFs specified by the normal 

AUG and a PTC in exon 1 by 10 codons can render a NMD-resistant transcript NMD-sensitive 

and vice versa. Surprisingly, the position of these insertions or deletions displays differential 

effects on both reinitiation- and NMD-efficiency of not only exon 1 nonsense mutations, but 

also of a normally reinitiation-incompetent and NMD-sensitive nonsense mutation in exon 2. 

54

JONATHAN DINMAN 

55 


mRNA destabilization by programmed ribosomal frameshifting and its effects 

on telomere maintenance and aging in yeast 

Jonathan Dinman 1, Ashton Belew 1, Rasa Rakauskaite 1, Olaf Haubenreisser 2, Jia Zhou 2, 

Lore Breitenbach-Koller 2 

1 University of Maryland, United States of America 

2 University of Salzburg, Austria 

Cis-acting mRNA elements that program ribosomes to shift translational reading frame were 

first discovered in viruses. Typical programmed -1 ribosomal frameshift (-1 PRF) signals are 

composed of a heptameric "slippery site" followed by an mRNA pseudoknot secondary 

structure. We previously developed a computational method to identify putative -1 PRF signals 

in eukaryotic genomic sequences (Belew et al., 2008), and candidate sequences were shown 

to stimulate significant -1 PRF (Jacobs et al., 2007). Analysis of these signals shows that 

ribosomes are directed to translate premature termination codons following a frameshift event, 

suggesting that -1 PRF signals can function as mRNA destabilizing elements via the 

nonsense-mediated mRNA decay (NMD) pathway. In addition, ribosome stalling induced by 

strong mRNA structures can promote mRNA degradation via 'No-go decay’ (NGD). Northern 

blot analysis using PGK1-based reporters containing -1 PRF signals from 4 genes showed that 

-1 PRF signals can promote significant mRNA destabilization through both pathways in yeast. 

Followup experiments on EST2 (telomerase) showed that NMD is the dominant pathway 

destabilizing this mRNA. Ablation of -1 PRF signals stabilizes the full length EST2 mRNA, 

affects telomere length, increases cell lifespan, and results in a mitotic delay.



SEVIM OZGUR 

Human Pat1 Controls the Assembly of Processing-Bodies and Promotes 

mRNA Degradation 

Sevim Ozgur, Georg Stoecklin 

German Cancer Research Center, Germany 

Processing (P)-bodies are small cytoplasmic foci that contain many enzymes required for 

mRNA deadenylation, decapping and decay. mRNAs destined for degradation are recruited to 

P-bodies where decay is thought to happen. In addition, P-bodies contain components of the 

RNAi-induced silencing complex such as argonaute proteins, miRNAs and translationally 

repressed mRNAs. Thus, P-bodies appear to serve a dual function: to transiently store mRNAs 

that are translationally silenced, and to target mRNAs for degradation by the decapping/5'-3' 

decay pathway. In yeast, the Pat1 protein was previously identified as an enhancer of 

decapping and mRNA decay. In addition, yeast Pat1 together with the helicase Dhh1/Rck 

localizes to P-bodies and acts as an inhibitor of translation during glucose starvation. We 

identified two possible human orthologs, hPat1 and hPatR, which share a recognizably 

conserved region of only 36 amino acid with yeast Pat1. We found that hPat1 localizes to 

P-bodies, whereas hPatR does not. Overexpressed hPat1 strongly increases the number of 

P-bodies, while its knock down suppresses P-bodies. In agreement with its localization, we 

found that hPat1 interacts with several P-body proteins. An acidic domain within hPat1 

interacts with the RNA helicase Rck, and the C-terminal region interacts with the 

exoribonuclease Xrn1 and the decapping enhancers Hedls, Lsm1 and Lsm4. We further show 

that an N-terminal region contains an aggregation-prone domain, whereas the acidic domain 

controls the step by which small aggregates grow into larger P-bodies. Using a tethering 

assay, we demonstrate that human Pat1 promotes the rapid degradation of mRNAs, whereas 

it does not appear to affect translation. Taken together, our data suggest that hPat1 plays an 

important role in controlling the assembly of P-bodies. In addition, hPat1 may promote rapid 

mRNA degradation by bridging subcomplexes that are important for the different steps of 

mRNA decay. 

56

FELIX TRITSCHLER 

57 


A metazoan-specific DCP1 C-terminal extension is required for the assembly 

of active mRNA decapping complexes in human cells 

Jörg Braun, Carina Motz, Gabrielle Haas, Vincent Truffault, Oliver Weichenrieder, Elisa 

Izaurralde, Felix Tritschler, Cátia Igreja 


The removal of the 5′ cap structure by the decapping enzyme DCP2 irreversibly commits 

eukaryotic mRNAs to degradation. DCP2 activity is stimulated by DCP1, and in multicellular 

eukaryotes this requires additional proteins including EDC4 (enhancer of decapping 4), which 

stabilizes the DCP1-DCP2 interaction. DCP2 is conserved, but its activation has mainly been 

studied in S. cerevisiae. However, in fungi EDC4 is absent and other decapping activators lack 

the extensions and additional domains present in metazoan proteins. In particular, DCP1 

consists of an N-terminal EVH1 domain conserved in all eukaryotes and a C-terminal extension 

that is only present in orthologs from metazoa and plants. Here, we show that this C-terminal 

extension is required for human DCP1a to be incorporated into active decapping complexes. 

These minimally contain DCP2 and EDC4 in addition to DCP1 and can assemble 

independently from alternative complexes comprising DCP1, EDC3 and the DEAD-box protein 

DDX6. Our results suggest the decapping network in multicellular eukaryotes has evolved an 

increased complexity that may provide additional opportunities for regulating mRNA 

degradation in these organisms.



MARKUS LANDTHALER 

PURE-CLIP - Transcriptome-wide identification of RNA targets and binding 

sites of RNA-binding proteins 

Mohsen Khorshid 1, Markus Landthaler 2, Lukas Burger 1, Mihaela Zavolan 1, Markus Hafner 3, 

Andrea Rothballer 3, Manuel Ascano 3, Anna-Carina Jungkamp 3, Mathias Munschauer 3, 

Alexander Ulrich 3, Scott Dewell 3, Thomas Tuschl 3 

1 Biozentrum der Universität Basel, Switzerland 

2 BIMSB, Germany 

3 Rockefeller University, United States of America 

RNA transcripts are subject to posttranscriptional gene regulation involving RNAbinding 

proteins (RBPs) that modulate stability, transport, editing and translation. A prerequisite for the 

understanding of these regulatory mechanisms is the generation of high-resolution maps of 

cellular protein-RNA interactions. To define protein-RNA networks, we developed a new 

method that allows for transcriptome-wide identification of the precise RNA recognition sites of 

regulatory RNA-binding proteins, which we refer to as Photoreactive-Uridine-Enhanced 

Crosslinking and Immunoprecipitation or PURE-CLIP. 4-Thiouridine, when provided to cultured 

cells, is incorporated into nascent RNAs and crosslinked to RNA-interacting proteins. In 

contrast to other methods, the sites of crosslinking are identified by mapping T to C transitions 

residing in the cDNA of libraries prepared from RNAs crosslinked to a specific RNA-binding 

protein. In human embryonic kidney cells, we mapped thousands of binding sites for the RBPs, 

Pumilio 2 (PUM2), Quaking (QKI), and the three members of the IGF2 mRNA-binding protein 

family (IGF2BP1, 2, 3), and deduced RNA motifs required for protein recognition. Knockdowns 

of the endogenously expressed RBPs indicate that QKI destabilizes its bound transcripts while 

IGF2BP1-3 stabilize their mRNA targets, indicating that our versatile approach to map 

RNA-protein interactions identified functional binding sites. 

58

JAMIE CATE 

Insights into protein synthesis from structures of the E. coli ribosome 

Jamie Cate, Jack Dunkle, Wen Zhang 


59 


Protein biosynthesis requires many large-scale rearrangements in the ribosome as each amino 

acid is added to a growing polypeptide chain. A key conformational change in the ribosome 

that is essential for translation is rotation of the small ribosomal subunit relative to the large 

subunit. Rotation of the ribosomal subunits occurs in all stages of translation–initiation, 

elongation, termination, and ribosome recycling–and is targeted by clinically useful antibiotics. 

In addition to rotation of the two ribosomal subunits, large-scale movements of the head 

domain of the small ribosomal subunit are thought to control the position of tRNAs as they 

traverse the three tRNA binding sites within the ribosome. As with subunit rotation, movement 

of the head domain is a target for clinically useful antibiotics. I will present new structures of the 

70S ribosome from Escherichia coli that reveal new aspects of the molecular basis for subunit 

rotation and movement of the head domain of the small subunit. In addition, I will present 

structures of the ribosome with antibiotics that shed light on how these compounds inhibit 

specific steps of the translocation mechanism.



STEFANO MARZI 

A dynamic view of translation initiation and its control by structured mRNAs in 

bacteria 

Stefano Marzi 1, Pierre Fetcher 1, Pascale Romby 1, Mathias Springer 2, Bruno Klaholz 3 

1 IBMC Strasbourg, France 

2 IBPC Paris, France 

3 IGBMC Illkirch, France 

Binding of the mRNA to the 30S subunit is one of the most critical steps of the translation 

initiation process in bacteria. mRNA structures directly influence this process and modulate 

protein synthesis. It is now admit that structured mRNAs bind to the 30S into two defined 

steps: docking of the mRNA followed by an accommodation step to promote the correct 

codon-anticodon interaction into the P site. The docking step, commonly referred to as the 

pre-initiation step, is rapid and transient while the accommodation step can be influenced by 

the stability of the structures that very often need to be melted. Recently, we have shown by 

cryo-EM, how mRNA folded elements are recognized by the platform of the 30S before the 

mRNA is adapted into the decoding channel1. These cryo-EM structures have shown that the 

pre-accommodated mRNA interacts with several ribosomal proteins located at the platform 

(S2, S7, S11, S18 and presumably S1). A systematic structure and sequence analysis revealed 

that conserved residues of these proteins form patches of positive charges on the surface of 

the platform suggesting their implication in binding structured mRNA on the ribosome. We 

have recently performed mutations at strategic positions of these r- proteins to analyze their 

effect in vivo on translation efficiency and its control, and in vitro on the formation of the 

initiation complexes involving structured mRNAs. These experiments will address the 

contribution of the conserved residues of the ribosomal proteins in docking folded mRNAs or in 

promoting mRNA adaptation and the intrinsic properties of initiating 30S to melt these 

structures. Our main objective is to get a dynamic view of the different steps leading to a 

productive initiation complex involving structured mRNAs. 

1. Marzi, S. et al. (2007) Cell, 130:1019-31. 

60

CLAUDIO GUALERZI 

61 


Mechanism of ribosomal recruitment of fMet-tRNA by bacterial translation 

initiation factor IF2 

Marcello Carotti 2 Andrey Konewega 1, Pohl Milon 1, Wolfgang Wintermeyer 1, 

Marina Rodnina 1, Claudio Gualerzi 2 

1 MPI for Biophysical Chemistry, Germany 

2 University of Camerino, Italy 

The recognition of fMet-tRNA by IF2 and a potential analogy between this factor and e/aIF2 

and EF-Tu and EF1 have contributed to attributing the role of “fMet-tRNA carrier” to IF2. In the 

absence of the 30S subunit, IF2 can form a fairly stable (Kd ~ 1 μM) ternary complex (TC) with 

fMet-tRNA and GTP. Thus, the concentrations of these ligands would theoretically allow the 

formation of such a TC in vivo. To test if fMet-tRNA is indeed carried to the 30S by IF2, the 

interactions between fMet-tRNA, IF2 and 30S subunit have been analyzed by fast filtration and 

FRET-based rapid kinetic methods. We found that IF2 binds to the 30S through a fast and a 

slow step. The first step is both guanosine nucleotide- and IF1-independent while the second 

step, which does not display a linear dependence on the IF2 concentration, depends upon the 

presence of IF1. Based on what is known concerning the structure of IF2 and the functional 

properties of its domains, it is likely that the fast step is due to the interaction of the 30S with 

the N-terminal “ribosomal anchor” of IF2 while the slower step reflects the interaction of its 

“functional core” (subdomains G2 and G3) and may entail an IF1-promoted accommodation of 

IF2 on the 30S surface. Compared to IF2, fMet-tRNA binds to the 30S complex ~ 20 times 

slower and its apparent binding rate depends linearly on its concentration, indicating the 

occurrence of a two-components binding reaction. Furthermore, fMet-tRNA binding is not 

faster when the initiator tRNA is pre-incubated with IF2 and GTP to form a TC. Instead, under 

these conditions fMet-tRNA binding is rate-limited by the accommodation of IF2 on the 30S, 

while IF2 binding is also slowed down and its binding may be rate-limited by the dissociation of 

the preformed TC. These observations indicate that, even though an IF2-GTP-fMet-tRNA TC 

may form, this is not productive and may result in a slower formation of the 30S initiation 

complex.



JOACHIM FRANK 

Structural Discrimination Between Cognate and Near-cognate Ternary 

Complexes By the Ribosome 

Leonardo Trabuco 1, Klaus Schulten 1, Xabier Agirrezabala 2, Jianlin Lei 2, Joachim Frank 3, 

Rodrigo F. Ortiz-Meoz 4, Rachel Green 4 

1 Beckman Institute, United States of America 

2 Columbia University, United States of America 

3 HHMI, Columbia University, United States of America 

4 HHMI, Johns Hopkins University, United States of America 

In this work, we investigate the structural basis of the induced-fit mechanism of the decoding 

process. We prepared cryo-EM maps from ribosomes programmed with near-cognate 

(UGA/stop) codons, loaded with initiation fMet-tRNAfMet in the P site, that were incubated with 

ternary Trp-tRNATrp•EF-Tu•GTP complexes in the presence of kirromycin. Using 

classification of 350,000 images, 3D reconstruction and molecular dynamics flexible fitting 

(MDFF), we find the majority (92%) of the ribosomes unbound, but a subpopulation (~8%) ) 

with the ternary complex bound. Here the near-cognate aa-tRNA is kinked as previously 

observed (Valle et al., 2003, Nature Struct. Biol. 10 , 899-906) but there are striking 

differences: (a) the distortion is distinct from the distortion seen in the cognate case, (b) the 

interaction between aa-tRNA and EF-Tu is changed, and (c) the switch I region of EF-Tu is 

apparently remodeled. Our results show that in the absence of the cognate aa-tRNA species, 

the cycling through the near-cognate species creates a steady state reflected by a small 

sub-population in which the ternary complex is arrested on the ribosome. Analysis of the 

interaction between aa-tRNA and EF-Tu and comparison with the cognate case may offer 

clues on how the conformational signals conveyed by the aa-tRNA to EF-Tu differ in the two 

cases. 

62

LASSE JENNER 

Crystal structure of the ribosome containing three tRNAs 

Lasse Jenner 1, Natalia Demeshkina 2, Gulnara Yusupova 2, Marat Yusupov 2 

1 CERBM-GIE / IGBMC, France 

2 IGBMC, Strasbourg, France 

63 


We have determined the X-ray crystal structure of a 70S ribosome functional complex at the 

state of elongation at 3.1A of resolution. This complex contains a 66 nucleotide mRNA and 

tRNAPhe in all three binding sites. The structure shows how the hypermodified nucleotide at 

position 37 of tRNAPhe augments P-site tRNA / mRNA interactions to prevent peptidyl-tRNA 

slippage and thereby frameshifting. Furthermore structures of vacant ribosomes and a 

ribosome initiation complex have been determined at high resolution. A detailed comparison of 

these structures will be presented in terms of conformational differences, and the importance 

of a novel intersubunit bridge will be discussed.



REBECCA VOORHEES 

Insights into substrate stabilization from structural studies of the peptidyl 

transferase center of the intact 70S ribosome 

Rebecca Voorhees, Weixlbaumer Albert, David Loakes, Ann Kelley, V Ramakrishnan 

Medical Research Council: Laboratory of Molecular Biology, United Kingdom 

No high-resolution structure of the intact ribosome has contained a complete active site 

including both A- and P-site tRNAs. There has also been disagreement as to the validity of 

studies performed on the 50S subunit alone, as well as the relevance of the small 

oligonucleotide tRNA-mimics used in structural studies of the PTC. Additionally, though 

structures of the 50S subunit found no ordered proteins at the PTC, biochemical evidence 

suggests specific ribosomal proteins are capable of interacting with the 3' ends of the tRNA 

ligands. In particular, data suggests that ribosomal proteins L16 and L27 may be closer to the 

active site than previously reported and may therefore play a supporting role in catalysis by the 

ribosome. However no structural data have yet elucidated the possible role of these proteins 

in peptidyl transfer. 

Here we present structures at 3.6 A and 3.5 A resolution of the 70S ribosome in complex with 

A- and P-site tRNAs that mimic pre- and post-peptidyl transfer states. Both structures contain 

a complete peptidyl transferase center including an A-site tRNA for which the CCA and amino 

acid at its 3' end are ordered within the PTC. Together, these structures provide a more 

complete description of the ligand-bound ribosome and demonstrate that the PTC is very 

similar between the 50S subunit and the intact ribosome. Furthermore, density for the 

N-terminal tail of L27 as well as that for protein L16 were clearly resolved, thus revealing their 

interactions with the ribosomal substrates. These interactions provide insights into the role of 

these proteins in stabilizing the tRNA substrates and facilitating peptidyl transfer by the 

ribosome. Finally, the structures should dispel concerns about the validity of previous 

structural work on the PTC using the 50S subunit. Further work on other functional complexes 

will also be discussed. 

64

NIELS FISCHER 

The trajectory of tRNA movement through the ribosome visualized by 

time-resolved electron cryomicroscopy 

65 


Niels Fischer, Wolfgang Wintermeyer, Holger Stark, Andrey L. Konevega, Marina Rodnina 

Max Planck Institute for Biophysical Chemistry, Germany 

During the translocation step of protein synthesis, a complex of two tRNA molecules bound to 

mRNA moves through the ribosome. The reaction is intrinsically spontaneous and reversible 

but is greatly accelerated and driven in the forward direction by the elongation factor G at the 

cost of GTP hydrolysis. Here we report the trajectory of the spontaneous tRNA movement 

through the ribosome derived from a set of ~50 cryo-EM reconstructions. Image datasets were 

collected at different time points of tRNA movement and computationally separated into 

homogeneous sub-populations of ribosome complexes. We identify a number of pre- and 

posttranslocation substates of the ribosome that differ in the positions of the tRNAs, relative 

arrangement of the 30S and 50S subunits and the conformation of the 30S subunit. 

Comparing the distribution of the substates at the early, intermediate and late states of the 

reaction allowed us to simulate the trajectory of tRNA motion through the ribosome and reveal 

the coupling between the tRNA movement and the conformation changes of the ribosome.



CHRISTIAN SPAHN 

Visualization of conformational modes of ribosomal complexes by multi-particle 

cryo-EM 

Charite - Universitätsmedizin Berlin, Germany 

The ribosome is a dynamic macromolecular machine capable of undergoing large-scale 

conformational changes. Throughout the four functional phases of translation, i.e. initiation, 

elongation, termination and recycling the ribosome is controlled and guided by translation 

factors and binding of such a factor can induce conformational movements within the 

ribosomal machinery. However, the ribosome appears to be also capable of spontaneous 

conformational changes that occur in the absence of factor binding. Recent structural work 

has shown that pre-translocational (PRE) complexes from prokaryotes are conformational 

heterogeneous and that the ratchet-like subunit rearrangement and tRNA hybrid state 

formation occurs in a subset of the complexes [1, 2]. This behaviour has been previously 

proposed by single molecule FRET experiments [3, 4]. By using cryo-EM and multi particle 

methods we can directly visualize various conformational modes of the ribosome in a variety of 

other ribosomal complexes as well, including a PRE complex from eukaryotes, a decoding 

complex [5], a 80S-IRES complex and complexes between the 70S ribosome and EF-G. Thus, 

the ribosome is even more dynamic than previously anticipated. Our results support the 

emerging theory of the energy landscape for the ribosome [4]. 

[1] X. Agirrezabala et al., (2008) Mol. Cell 32: 190-197 

[2] P. Julián et al., (2008) PNAS 105: 16924-16927 

[3] R.A. Marshall et al., (2008) Annu. Rev. Biochem. 77:177-203 

[4] J.P. Munro et al., (2009) Trends Biochem. Sci., in press 

[5] J.C. Schuette (2009) <strong>EMBO</strong> J. 28:755-765 

[6] M. Schüler et al., (2006) Nature Struct. Mol. Biol. 13:1092-1096 

66

REBECCA KOHLER 

67 


YidC and Oxa1 Form Dimeric Insertion Pores on the Translating Ribosome 

Christiane Schaffitzel 1, Rebecca Kohler 2, Daniel Boehringer 2, Basil Greber 2, Nenad Ban 2, 

Rouven Bingel-Erlenmeyer 3, Ian Collinson 4 

1 EMBL Grenoble, France 

2 ETH Zuerich, Switzerland 

3 Swiss Light Source, Switzerland 

4 University of Bristol, United Kingdom 

The YidC/Oxa1/Alb3 family of membrane proteins facilitates the insertion and assembly of 

membrane proteins in bacteria, mitochondria and chloroplasts. The importance of the 

YidC/Oxa1/Alb3 proteins is reflected by their extraordinary functional complementarity, which 

spans large evolutionary distances. Here, we show that Escherichia coli YidC and 

Saccharomyces cerevisiae Oxa1 interact directly with the bacterial ribosome. We also present 

the structures of both YidC and Oxa1 bound to E. coli ribosome nascent chain complexes 

determined by cryo-electron microscopy. Dimers of YidC and Oxa1 are localized above the exit 

of the ribosomal tunnel. Crosslinking experiments show that the ribosome specifically stabilizes 

the dimeric state of the two “insertases”. Functionally important and conserved 

transmembrane helices of YidC and Oxa1 were localized at the dimer interface by cysteine 

crosslinking. Both Oxa1 and YidC dimers contact the ribosome at ribosomal protein L23 and 

conserved rRNA helices 59 and 24 similarly to what was observed for the non-homologous 

SecYEG translocon. We suggest that dimers of the YidC and Oxa1 proteins form insertion 

pores for nascent transmembrane proteins and share a common overall architecture with the 

non-homologous SecY monomer. 

Kohler, R., Boehringer, D., Greber, B., Bingel-Erlenmeyer, R., Collinson, C., Schaffitzel, C., 

Ban, N. (2009). YidC and Oxa1 form dimeric insertion pores on the ribosome. Molecular Cell 

34, 344-53.



TILMANN ACHSEL 

Identification and characterisation of the human Pat1: a novel deadenylation 

factor 

Antonio Totaro 1, Claudia Mattioli 1, Monika Raabe 2, Henning Urlaub 2, Tilmann Achsel 3, 

Giorgio La Fata 3 

1 Fondazione Santa Lucia, Rome, Italy 

2 Max Planck-Institut Göttingen, Germany 

3 VIB - K.U. Leuven, Belgium 

The LSm–Pat1p complex has an important role in yeast mRNA degradation, as it is required 

for translational shutdown after deadenylation. While the LSm proteins are highly conserved 

throughout evolution, there is no obvious Pat1 homologue in vertebrates. We developed a 

novel immunoprecipitation method that yields virtually pure complexes and thus limits, after 

mass spec analysis, the tedious screening of many potentially false positives. In HeLa 

cytoplasmic extracts, the method identified just one non-LSm protein that specifically 

co-purifies with LSm1. The interaction with LSm1 was verified by conventional 

immunoprecipitation. As the protein shares homology with the central domain of yPat1p, we 

name it hPat1. As expected, the protein localizes to the P bodies, and its N-terminus contains 

an aggregation domain typical of the nucleators of P bodies. When tethered to a reporter 

mRNA, hPat1 reduces protein expression, consistent with being a functional orthologue of 

yPat1p. Surprisingly, however, the reduction is owed primarily not to translational repression 

but to deadenylation, indicating that hPat1 acts further upstream in the mRNA decay cascade 

than its yeast counterpart. Depletion of endogenous hPat1 stabilises only one of the three 

ARE-containing mRNAs that we tested by reducing the portion of the transcript with critically 

short poly(A) tails. hPat1 thus is a novel deadenylation factor that specifically regulates a subset 

of the mRNAs. 

68

RASHID AKBERGENOV 

69 

Poster Abstracts 

rRNA Sequence Polymorphism within the Bacterial Domain and Susceptibility 

to Drugs Targeting Protein Synthesis 

A. Subramanian, S. Kalapala, M. Bertea, M. Kulstrunk, Erik Böttger, Rashid Akbergenov 

Institut für Medizinische Mikrobiologie, Universität Zürich, Switzerland 

The ribosome is target for many antibacterial agents, e.g. aminoglycosides, tetracyclines, 

macrolides, ketolides, lincosamides, oxazolidinones. It is unclear whether the in part significant 

polymorphism present in a drug’s binding site affects antibiotic susceptibility or resistance 

development. To address this question we have investigated the drug binding pocket of 

aminoglycosides and macrolide/ketolides. The drug binding site of aminoglycosides maps to 

helix 44 of the 16S rRNA (A-site), while that of macrolides/ketolides localizes to domain V of 

23S rRNA (peptidyl-transferase center). Sequence polymorphism within the aminoglycoside 

binding pocket involves 16S rRNA residues 1409-1491 and 1410-1490, polymorphism of the 

macrolide binding site involves 23S rRNA residues 2057-2611. Sequence alterations 

corresponding to the polymorphic residues identified were introduced by genetic means into a 

single rRNA allelic model to result in isogenic recombinants carrying homogenous populations 

of the mutated ribosomes under study. Subsequent drug susceptibility studies allowed for the 

following conclusions. 1. Natural sequence variations in the small subunit’s A-site of bacteria in 

part influence aminoglycoside susceptibility, but do not affect the resistance phenotype of the 

A1408G mutation. 2. Natural sequence variations in the bacterial large subunit’s 

peptidyl-transfer center do not affect macrolide/ketolide susceptibility, but significantly impact 

on the resistance phenotype of the A2058G mutation.



ROSS ANDERSON 

Investigating the Expression and Functions of the Poly(A)-Binding Protein 

Family Within Mammalian Gonads 

Nicola Gray , Lora McCracken, Ross Anderson, Matthew Brook 

MRC Human Reproductive Sciences Unit, University of Edinburgh, United Kingdom 

Translational regulation during gametogenesis frequently depends on changes in poly(A) tail 

length which occur in the cytoplasm. The function of these poly(A) tails is mediated by a family 

of poly(A)-binding proteins (PABP's). PABP's are multi-functional translation initiation factors 

that also have important roles in mRNA stability and non-sense mediated decay(1). 

To date, only the functions of the prototypical member of this family (PABP1) have been 

described in much detail. However, studies in Xenopus oocytes have established that other 

PABP's (e.g. ePABP) share the ability to promote translation(2). Thus, the presence of multiple 

structurally similar PABP's within the same cell(1) raises questions pertaining to their individual 

functions. 

Mammals encode four structurally related PABP proteins; PABP1, PABP4, tPABP, ePABP, 

and a structurally distinct protein, PABP5. The expression patterns of these proteins have not 

been studied in any detail in mammals, however, PABP1 and PABP4 are generally considered 

to be ubiquitously expressed. tPABP expression is limited to a subset of male germ cells, 

while ePABP appears to be present in germ cells and early embryos. 

Here, we undertake a detailed analysis of the expression patterns of the PABP family within the 

mammalian gonad and find that PABP proteins display complex overlapping yet distinct 

patterns of expression at both the RNA and protein level. A variety of biochemical and 

molecular assays have been employed to dissect the functions of different PABP's to explore 

the extent to which they have distinct molecular roles. 

1. Gorgoni B., Gray N.K. The roles of cytoplasmic poly(A)-binding proteins in regulating gene 

expression: a developmental perspective. Brief Funct Genomic Proteomic. 3(2):125-41 (2004) 

2. Wilkie G.S., Gautier P., Lawson D., Gray N.K. Embryonic poly(A)-binding protein stimulates 

translation in germ cells. Mol Cell Biol. 25(5):2060-71 (2005) 

70

DMITRY ANDREEV 

71 


Translation machinery can efficiently scan through the highly structured 5’ 

UTR of Apaf-1 mRNA containing putative IRES 

Dmitry Andreev, Sergey Dmitriev, Ilya Terenin, Ivan Shatsky 

Moscow State University, Russian Federation 

It is widely assumed that the secondary structure of 5’ UTR is a key feature which affects the 

efficiency of cap-dependent translation. Thus for long and highly structured 5’ UTRs alternative 

mechanisms, such as internal ribosomal entry, have been proposed. 

Here we demonstrate that the m7G-capped reporter mRNA with the 577 nt long 5’ UTR of 

Apaf-1 mRNA can be translated both in vivo and in vitro (in nuclease-untreated cytoplasmic 

extract) with efficiency comparable to that of 53 nt long 5’ UTR of β-globin, in spite of the fact 

that this leader contains several highly structured domains which are believed to constitute an 

IRES. It should be noted that placement of the Apaf-1 5’ UTR in the intercistronic position of a 

bicistronic mRNA results in an extremely weak translation of the second cistron as compared 

with the respective monocistronic construct. When m7G-cap is replaced by non-functional 

A-cap for monocistronic mRNAs, the translation driven by the Apaf-1 5’ UTR is strongly 

reduced. Insertion of uAUG codons in the 5’ UTR of Apaf-1 results in a dramatic reduction of 

translation not only for m7G-capped, but also for A-capped monocistronic mRNAs. 

Consecutive deletions of the structural domains in the Apaf-1 leader shows only modest 

changes in translation, in case of both m7G- or A-capped mRNAs. Thus, these data strongly 

suggest that the 40S ribosome is capable of efficient scanning the 5’ UTR of Apaf-1 over its 

entire length and this is the case for both capped and uncapped RNAs. However, the intriguing 

fact found is that in some cell lines the Apaf-1 5’ UTR is nevertheless more resistant (5-10 fold) 

to omission of the cap than other cellular 5’ UTRs used in this study. The mechanism which 

may determine the different cap-dependence of translation for various 5’ UTRs and which is 

alternative to the concept of cellular IRESs will be discussed.



JOSHUA ARRIBERE 

A comprehensive analysis of environmentally regulated yeast 5'UTR variants: 

annotation and insights into functionality in translation regulation 

Joshua Arribere, Maria F. Rojas Duran, Wendy V. Gilbert 

MIT - Gilbert Lab, United States of America 

5’UTR features can have large, condition-dependent effects on the translational efficiency of 

mRNAs. For example, uORFs in the 5’UTR of GCN4 allow selective translational activation of 

GCN4 when global translation is down-regulated by reduction of ternary complex via increased 

eIF2alpha phosphorylation. Similarly, IRES elements in some 5’UTRs permit selective 

cap-independent translation in glucose-starved cells when global translation is repressed by 

the decapping machinery. Alternative 5’UTR production is ubiquitous in eukaryotes and has 

enormous though under-appreciated regulatory potential. Although current evidence from 

cDNA sequencing suggests widespread 5’UTR heterogeneity in yeast, the full extent of 

variability of 5’UTRs within genes and across the genome is not known. We are employing a 

novel “cap-trap” protocol to sequence a comprehensive library of yeast 5’UTR variants 

expressed under three different growth conditions: rich media, amino-acid starvation, and 

glucose-starvation. Recent advances in deep-sequencing technology will allow us to 

confidently measure 5’UTR isoform distributions of even lowly expressed regulatory genes. The 

precise definition of yeast 5’UTRs is a critical step in the study of translation, particularly for the 

identification of cis-regulatory elements involved in ‘global’ and selective translational control. 

Since eukaryotic translation mechanisms are highly conserved, our work will likely provide 

insight into common regulatory themes attributable to 5’UTR heterogeneity. 

72

MICHELLE BADURA 

73 


DNA damage mediates a novel control of translation by signaling through the 

DNA damage response complex to control mTOR activity and 4E - BP1 

stability 


Ionizing radiation (IR) is a physiologically important stress to which cells respond by activation 

of multiple signaling pathways. Using immortalized and transformed breast epithelial cells, we 

demonstrate that IR regulation of translation occurs in non-transformed cells and is lost with 

transformation. In non-transformed cells, IR rapidly activates the MAP kinases ERK-1/2, 

resulting in an early transient increase in cap-dependent mRNA translation involving mTOR. 

This increase is radio-protective, enhancing translation of a subset of mRNAs encoding 

proteins involved in DNA repair and cell survival. Following the increase in translation, 

IR-sensitive (non-transformed) cells inhibit cap-dependent protein synthesis through a 

mechanism involving activation of p53, induction of Sestrin1 and 2, and stimulation of AMP 

kinase, resulting in inhibition of mTOR and hypo-phosphorylation of 4E-BP1. IR, but not other 

stresses such as hypoxia, is shown to inhibit proteasome-mediated decay of 4E-BP1, 

increasing its abundance and sequestration of eIF4E. In addition, over-expression of 4E-BP1 in 

transformed cells restores their ability to inhibit translation following IR, indicating the 

importance of translational control in response to DNA damage. The IR-response signal that 

controls mTOR-dependent translation is shown to be assembly of the DNA damage response 

machinery, consisting of Mre11, Rad50 and NBS1 (MRN), activation of ATM, and activation of 

p53. These results link genotoxic signaling from the DNA damage response complex to the 

control of protein synthesis.



VIDYA BALAGOPAL 

Stm1 modulates translation and mRNA decay in Saccharomyces cerevisiae 

University of Arizona, United States of America 

The control of mRNA degradation and translation are important for the regulation of gene 

expression. mRNA degradation is often initiated by deadenylation, which leads to decapping 

and 5' to 3' decay. In the budding yeast Saccharomyces cerevisae, decapping is promoted by 

Dhh1 and Pat1 which both inhibit translation initiation and promote decapping. To understand 

the function of these factors, we identified the ribosome binding protein Stm1, as a multicopy 

suppressor of the temperature sensitivity of the pat1Δ strain. Stm1 loss of function alleles and 

over-expression strains show several genetic interactions with Pat1 and Dhh1 alleles in a 

manner consistent with Stm1 working upstream of Dhh1 to promote Dhh1 function. Consistent 

with Stm1 affecting Dhh1 function, stm1Δ strains are defective in the degradation of the EDC1 

and COX17 mRNAs, whose decay is strongly affected by the loss of Dhh1. In vitro, 

recombinant Stm1 inhibits translation and leads to the accumulation of a novel mRNP 

suggesting Stm1 inhibits a specific step in translation. We interpret these results to suggest 

that Stm1 induced translationaly stalled mRNPs are remodeled by the DEAD-box helicase, 

Dhh1, to allow the mRNA to be targeted for translation repression, decapping, and/or 

potentially to re-enter translation. This could be a novel function for repression proteins/ 

granules as a quality control mechanism for translation. 

74

AMANDINE BASTIDE 

Investigation of Translational Regulation during Cold-Shock 

Anne Willis 1, Mark Smales 2, Amandine Bastide 1, Ruth Spriggs 1, Martin Bushell 1 

1 Nottingham University, United Kingdom 

2 University of Kent, United Kingdom 

75 


Many industrial fermentations for biotherapeutics production operate a biphasic temperature 

strategy: cells are initially cultured at 37°C to accumulate biomass and then the temperature is 

shifted to sub-physiological temperatures (typically 32°C) to enhance protein production and 

prolong culture viability in the stationary growth/production phase. However, as cells approach 

maximum concentrations and as a direct consequence of cold stress there is suppression of 

global translation initiation by the normal cap-dependent mechanism due to an increase in 

phosphorylation of the alpha subunit of eIF2. To identify those mRNA preferentially 

up-regulated at the translational level during cold-shock, we have performed polysomal mRNA 

profiling. This technique couples sucrose density gradient separation of polysomally 

associated mRNAs with cDNA microarray and allows a ‘translational profile’ to be obtained. 

This technique was used following culture of human embryonic kidney (HEK) cells at 32°C and 

the data obtained compared to cells cultured at normal temperature (37°C). mRNAs 

translationally up-/down-regulated during cold-shock were identified: those showing increased 

polysomal association following cold-shock encoded proteins with functions in cell cycle 

regulation and those associated with ER stress. Changes in micro RNA expression following 

cold-shock were also determined since these are known to permit selective up-regulation of 

mRNAs under other cell stresses. Data obtained from both screens were analysed to identify 

putative sequence elements in the 5’ and 3’ UTRs of the mRNAs translationally up-regulated 

following cold-shock. The role of these elements in translational regulation is under 

investigation. In the longer term these data will be used to design vectors with UTRs that 

confer and aid selective translation during cold-shock that are able to override the global 

shutdown of mRNA translation.



GRAHAM BELSHAM 

Themes and variations in HCV-like IRES elements 

Chanti Polacek 1, Thomas Bruun Rasmussen 1, Lisa Roberts 2, Margaret Willcocks 2, Graham 

Belsham 3 

1 DTU, Denmark 

2 Surrey University, United Kingdom 

3 Technical University of Denmark, Denmark 

The positive sense RNA genomes from all members of the picornaviridae (e.g. poliovirus, 

foot-and-mouth disease virus) and from certain flaviviridae (hepatitis C virus (HCV), classical 

swine fever virus, CSFV) contain IRES elements. There are now recognized to be 4 different 

classes of IRES element present within the picornavirus family. Surprisingly, one of these is 

closely related to the IRES present within HCV and the pestiviruses such as CSFV. These 

elements contain two major domains, termed domain II and domain III, the latter includes an 

essential pseudoknot (IIIf). Although the overall structure of the HCV and CSFV IRES elements 

is very similar there are key differences, for example the presence of domain IIId2 and the 

bipartite stem 1 of the pseudoknot in CSFV. The IRES elements from the picornaviruses PTV, 

AEV, PEV-8 and SV2 are most closely related to the HCV IRES. However, Seneca Valley virus 

(SVV), a recently identified picornavirus, has an IRES which is more closely related to the CSFV 

IRES and shares with it a requirement for about 50nt of coding sequence. Mutational analysis 

of the CSFV and SVV IRES elements has been performed. While modification of the 

pseudoknot regions is very deleterious for the activity of each of these IRES elements, the IIId2 

domain is rather insensitive to modification. Furthermore, although complete removal of 

domain II has a major effect on IRES function (80-90% loss of activity), the domain II 

sequences can be extensively modified and even completely replaced without dramatic loss of 

IRES activity when assayed in the context of dicistronic mRNAs within cells. 

76

ALEXEY BENYUMOV 

77 


Translational regulation of the epithelial – to - mesenchymal transitioninsights 

form mesoderm restriction in zebrafish gastrulation 

Alexey Benyumov, Mark Peterson, Vitaly Polunovsky, Peter Bitterman 

Department of Medicine, University of Minnesota, United States of America 

The epithelial-to-mesenchymal transition (EMT) is an essential differentiation program in early 

embryonic development when germ layers and organ topography are established¹. The 

process involves loss of both cell polarity and tight inter-cellular junctions as ectodermal 

epithelial cells acquire a non-polarized, migratory mesenchymal phenotype. However, in some 

of the most prevalent and morbid human diseases, the EMT is usurped to mediate 

pathological changes. In cancer the EMT enables epithelial cells to transit the cancer pathway 

acquiring the capacity to migrate, invade tissue planes and metastasize; in fibroproliferative 

diseases of the lung, liver and kidney, EMT is a source of pathological fibroblasts that deposit 

connective tissue distorting normal anatomy thus leading to organ failure. Zebrafish, a 

genetically tractable vertebrate species amenable to drug screening and cancer research, 

provides several models of EMT. We employed one such model – EMT in mesoderm formation 

– to study translational control of the EMT in vivo using polyribosome preparations to stratify 

mRNA according to the number of ribosomes bound. Tissue samples containing embryonic 

shield (destined to undergo EMT) and flanking tissue samples (no EMT) were obtained by 

microdissection from mid-gastrula blastoderms at 50-65% of epiboly. Polyribosome-stratified 

RNA was fractionated as previously described², and RNA from translationally active and 

inactive fractions was analyzed by Q-PCR for stage and linage-specific marker expression: 

EMT-relevant transcripts, ectoderm markers and housekeepers. Here we present pilot 

experimental data showing a pair-wise comparison of the translational activity of selected 

messages in shield (“EMT”) vs. flanking (“no-EMT”) samples. ¹ - Thiery JP and Sleeman JP. 

2006. Nat Rev Mol Cell Biol, 7, 131-142. ² - Larsson O, Perlman DM, Fan D. et al. 2006. 

Nucleic Acids Res, 34, 4375–86.



RUMPA BHATTACHARYA 

Regulation of mRNA transaltion during recovery from heat shock 

Rumpa Bhattacharya, Jnanankur Bag, Thangima Zannat 

University of Guelph, Canada 

The Poly (A) binding protein (PABP) plays a crucial role in regulating the translation and stability 

of eukaryotic mRNA. PABP behaves like an early response gene as its expression is controlled 

by growth stimulation. During exposure of cells to heat shock cap dependent translation of 

normal cellular mRNAs is repressed and IRES mediated translation of mRNAs encoding HSPs 

is induced. However, when cells are transferred to an ambient temperature recovery begins 

with translation of capped cellular mRNAs while expression of heat shock proteins continues. 

We found that in the heat-shocked cells PABP1 and eIF4G complex dissociates, and both 

polypeptides translocate with the HSP27 to the nucleus as detergent insoluble granules. 

During recovery after heat shock, PABP1 and eIF4G are redistributed into the cytoplasm and 

co-localized with each other. In addition, PABP1 expression is up-regulated by approximately 

2-3 folds and its translation efficiency increased during the recovery period. Results of our 

studies show that the terminal oligopyrimidine (TOP) cis-element of PABP1 mRNA is 

responsible for the preferential increase of PABP1 mRNA translation in cells undergoing 

recovery from heat shock. Since both HSP 70 and HSP 27 accumulate in large quantities in 

cells during the recovery period we investigated the role of HSP 70 in regulating TOP and IRES 

mediated translation. Cells stably transfected with a doxycycline inducible dicistronic HSP 70 

and GFP construct where the GFP expression was under the control of an IRES cis element 

showed that HSP 70 expression alone was sufficient for IRES mediated translation but 

induction of PABP expression required exposure of cells to heat shock. Translation of both 

cistrons continued for at least 48 hours at 37˚C. Our results suggest that cap and IRES 

dependent mRNA translation are not mutually exclusive processes. [Supported by NSERC] 

78

DANIEL BOEHRINGER 

Co-translational folding and membrane insertion of newly synthesized 

polypeptides 

Christiane Schaffitzel 1, Daniel Boehringer 2, Rebecca Kohler 2, Nenad Ban 2 

1 EMBL Grenoble, France 

2 ETH, Switzerland 

79 


Newly synthesized polypeptides are subjected to enzymatic processing, chaperone-assisted 

folding and targeting to translocation pores at membranes concurrently with their synthesis by 

the ribosome. The ribosome itself plays a key role in governing the interplay between the 

various factors involved. We have studied the interaction between ribosomes and various 

enzymes and factors involved in nascent chain processing, folding, membrane targeting and 

insertion. 

Trigger factor, the first protein to interact with the nascent chains, provides a protective cradle 

for folding of the newly synthesized polypeptide. We suggest that the growing nascent chain 

follows a defined path inside trigger factor and thereby the interaction with targeting factors, 

signal recognition factor, and processing factors, peptide deformylase and methionine 

aminopeptidase, could be coordinated. 

For the co-translational insertion of membrane proteins two different systems exists, the Sec 

system and the YidC system of membrane protein insertases. These insertase systems can act 

alone or together depending on the substrate protein. In the Sec dependent insertion nascent 

transmembrane segments are laterally released from SecY into the lipid bilayer. In this case 

YidC can interact with these nascent transmembrane segments acting like a membrane 

chaperone. In cases when YidC acts alone, we propose that it forms dimeric pore that can 

insert proteins mechanistically analogous to SecY. This requires different modes of interaction 

of YidC with the ribosome suggesting a dynamic model for the interplay of both insertase 

systems on the ribosome.



ANDREW BOTTLEY 

Translational profiling reveals an important role for eIF4A in the regulation of 

specific mRNAs 

Nicola Phillips, Thomas Webb, Keith Spriggs, Andrew Bottley 

Nottingham University, United Kingdom 

In eukaryotes, most translation initiation is coordinated by recruitment of the translational 

machinery to a cap structure at the 5' terminus of the message and represents a major target 

for direct, rapid control of the rate of translation.This initiation complex then scans through the 

5' UTR of the mRNA until it recognises an AUG initiation codon, at which point protein 

synthesis can begin. 

Ribosome scanning is an active process driven by the ATP dependent RNA helicase eIF4A, the 

prototypical DEAD-box helicase and most abundant translation initiation factor. The 5' UTR of 

many messages is long and structured and presents a significant physical barrier to scanning 

ribosomes. It has been proposed that the requirement of an mRNA for eIF4A in vitro is 

proportional to the degree of 5' UTR secondary structure. Inhibiting eIF4A may therefore not 

only reduce the level of proteins encoded by transcripts with long structured 5’UTRs, but may 

also result in an elevation of the level of proteins encoded by transcripts possessing shorter 

less complex UTRs. 

It has not yet been determined on a large scale which mRNAs are regulated by eIF4A activity. 

Therefore we have used a translational profiling approach to identify mRNAs that are 

particularly sensitive to eIF4A. By treating cells with the eIF4A inhibitor hippuristanol, isolating 

differentially labelling polysomal and subpolysomal pools of mRNA, and then hybridising 

probes derived from these pools to cDNA microarray chips, we have identified putative targets 

for regulation by translational control through eIF4A inhibition. Some eIF4A sensitive transcripts 

are associated with disease when elevated, while conversely some transcripts insensitive to 

eIF4A inhibition are cytoprotective. This suggests a mechanism by which a subset of mRNAs 

encoding regulatory proteins may be co-ordinately regulated and by extension may provide an 

attractive model for therapeutic intervention. 

80

SAVERIO BROGNA 

Visualization of ribosome subunits interaction in cells 

Saverio Brogna, Khalid Al-Jubran, Preethi Ramanathan 

University of Birmingham, United Kingdom 

81 


The 60S and 40S ribosome subunits are mostly assembled in the nucleolus but mRNA binding 

and 80S formation are believed to occur only in the cytoplasm during translation. To track 

ribosome subunits association we have tagged ribosomal proteins located near the subunits 

interface, with bimolecular fluorescence complementation (BiFC) fragments expected to fold 

into a functional fluorescent protein upon ribosome subunits association. Using this approach 

we were able to visualize ribosome subunits joining in living Drosophila cells, both in cell culture 

and in flies. As expected, most of the interaction occurs in the cytoplasm and is translation 

dependent. However, we also detected interaction in the nucleolus and, when blocking nuclear 

export, also in the nucleoplasm: suggesting that ribosome subunits can interact prior to 

nuclear export.



HANNAH BURGESS 

PABP1 and PABP4 relocalise to the nucleus following UV irradiation 

Nicola Gray 1, Hannah Burgess 2, Matthew Brook 2, Christine Salaun 3, Sheila V. Graham 3, 

Lyndsay Howard 3 

1 Medical Research Council, United Kingdom 

2 MRC HRSU / University of Edinburgh, United Kingdom 

3 University of Glasgow, United Kingdom 

Poly(A)-binding proteins (PABPs) are important regulators of mRNA translation and stability. In 

mice and humans five cytoplasmic PABPs with a common domain structure have been 

described - PABP1, tPABP, PABP4, PABP5 and ePABP. Work from our lab and others has 

demonstrated that a key difference between mammalian PABP proteins is their tissue and 

cellular distribution. The vast majority of research on PABP mechanism, function and 

sub-cellular localisation is however limited to PABP1. 

PABP1 is known to localise to cytoplasmic stress granules, sites of mRNA sorting and storage, 

following certain cellular stresses e.g. arsenite, osmotic shock. PABP1 has also been shown to 

localise to the nucleus during rotavirus infection, after heatshock and following treatment with 

transcriptional inhibitors such as actinomycin D. Here we show that PABP4 is also a 

component of arsenite-induced stress granules. Further, we show that both PABP1 and 

PABP4 relocalise to the nucleus following UV-C irradiation and a small number of additional 

stresses. We show that nuclear export of PABP1 and PABP4 is not dependent on the CRM-1 

pathway as PABP1 and PABP4 remain cytoplasmic upon treatment with leptomycin B, in 

contrast to paxillin, a classical NES containing protein which has been suggested to be 

responsible for the nuclear export of PABP1. We find that accumulation of PABP proteins in 

the nucleus is not coincident with transcriptional shut-off, suggesting that the transient 

transcriptional block which occurs during UV-stress is not linked to nuclear PABP 

accumulation. We are further investigating the mechanism of PABP nuclear accumulation, 

focusing on the links between poly(A) mRNA and PABP localisation. 

82

MARTIN BUSHELL 

Localisation of microRNA – repressed mRNAs to p-bodies prevents an 

initiation independent viral IRES from overcoming microRNA repression 

Anne Willis 1, Eric Jan 2, Martin Bushell 1, YiWen Kong 1, Ian Cannell 1 

1 Nottingham University, United Kingdom 

2 University of British Columbia, Canada 

83 


MicroRNA (miRNA) repression is mediated by a number of mechanisms, one of which is the 

direct inhibition of protein synthesis. Recently we have shown that transcription driven by 

specific promoters of the miRNA-targeted mRNA can dictate translational repression at either 

the initiation step or the post-initiation step. Specifically, SV40- and TK-initiated transcription 

leads to miRNA-dependent repression at initiation and post-initiation steps, respectively. To 

gain further mechanistic insight into this process, we have utilised the intergenic internal 

ribosome entry segment (IRES) of the Cricket Paralysis virus (CrPV), which engages translation 

in a factor-independent manner. We postulated that CrPV IRES-dependent translation should 

overcome the initiation block mediated by a miRNA-targeted mRNA transcribed by the SV40 

promoter. Rather surprisingly, miRNA-targeted reporter RNAs containing the CrPV IRES was 

unable to overcome this initiation block after transfection of the plasmid into cells. However, 

the CrPV IRES could overcome this repression when the reporter RNA was in vitro transcribed 

and transfected into cells, suggesting that the biogenesis of the CrPV IRES-containing 

transcript dictates whether miRNA-targeted RNAs are translationally repressed. These data 

suggests that SV40-derived transcripts may be compartmentalised away from ribosomes, 

possibly in P-bodies. In agreement with this, deletion of LSM family members (LSM1 and 3, 

essential for P-body formation), enabled the CrPV IRES to overcome microRNA-mediated 

repression. Our data therefore suggest that while P-bodies are not essential for translational 

repression by mRNAs, they have a subsequent role in preventing ribosomes from accessing 

repressed mRNAs. This raises interesting possibilities for the role of P-bodies in the 

microRNA-repression mechanism.



ANDERS BYSTRÖM 

Wobble uridine modifications in yeast 

Bo Huang 1, Marcus Johansson 1, Jian Lu 1, Anders Esberg 1, Anders Byström 2 

1 Molecular Biology, Sweden 

2 Umea University, Sweden 

Elongator complex consisting of the six Elp1-Elp6 proteins is conserved from yeast to humans. 

In yeast, Elongator has been proposed to participate in three distinct cellular processes; 

transcriptional elongation, polarized exocytosis, and formation of 5-methoxycarbonylmethyl 

(mcm5) and 5-carbamoylmethyl (ncm5) side-chains of wobble uridines in tRNA. We have 

shown that the phenotypes of Elongator deficient cells linking the complex to transcription and 

exocytosis are suppressed by increased expression of two tRNA species. Elongator is required 

for formation of the mcm5 group of the modified wobble nucleoside 

5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) in these tRNAs. Our results indicate that in 

cells with normal levels of these tRNAs, presence of mcm5s2U is crucial for 

post-transcriptional expression of gene products important in transcription and exocytosis. 

Furthermore we have shown that presence of mcm5 and ncm5 side-chains promote decoding 

of G-ending codons. 

84

IVÁN CAJIGAS 

Regulation of mRNA stability in the hippocampus 

Iván Cajigas 1, Erin Schuman 2 

1 California Institute of Technology, United States of America 

2 California Institute of Technology/ HHMI, United States of America 

85 


It is clear that de novo protein synthesis plays an important role in synaptic transmission and 

plasticity. A substantial amount of work has demonstrated that mRNA translation in the 

hippocampus is spatially controlled and that dendritic protein synthesis is required for different 

forms of long-term synaptic plasticity. Despite many recent observations that emphasize the 

importance of local protein synthesis in memory formation, little is known about the 

determinants that aid dendritic mRNA translation. In this work we are testing the hypothesis 

that neuronal activity regulates mRNA stability in order to facilitate local protein synthesis. Using 

confocal microscopy and immunofluorescence experiments we have gathered evidence 

showing that factors involved in modulating the half-life of mRNAs are present in dendrites and 

that their localization is regulated by neuronal activity. In order to obtain additional insights on 

the role of mRNA stabilization in the hippocampus, we are currently developing techniques to 

identify and track the localization of newly synthesized RNAs in neurons. Altogether these 

results suggest that mRNA stability can contribute to synaptic plasticity and memory 

processing.



CORNELIS CALKHOVEN 

Discovery of Translationally Active Small Molecules that Inhibit Proliferation of 

Cancer Cells Using Translation Re-initiation Index (TRI) Determination 

Cornelis Calkhoven, Anna Bremer, Christine Kamperdick, Matthias Görlach 

Leibniz Institute for Age Research - Fritz Lipmann Institute, Germany 

mRNAs coding for proliferation-promoting or survival factors often harbor uORFs as 

cis-regulatory elements that restrain their translation to conditions were re-initiation is favored. 

Interference with aberrantly enhanced translation re-initiation may suppress the expression of 

those factors, and could reduce the proliferation and survival potential of cancer cells. 

However, only few anti-cancer drugs are known to interfere with translation (re-)initiation, 

leaving its therapeutic potential largely unexplored. We developed cell lines containing 

uORF-regulated dual luciferase reporters that are designed for high-throughput screening 

strategies to facilitate the search for compounds that specifically inhibit or enhance translation 

re-initiation. Using these reporter cell lines we identified small molecules from a library of low 

molecular weight scaffolds (SHAPES library) that either decrease or increase the translation 

re-initiation indexed against regular translation initiation. In a follow-up screen we identified 

further modified small molecules with a low translation re-initiation index (TRI). These small 

molecules inhibit proliferation of anaplastic large cell lymphoma cell lines Karpas 299 (ALK+) 

and FE-PD (ALK-) that represent lymphomas with poor prognosis, where novel therapeutic 

options are needed. We show that a key event in the anti-proliferative action of the small 

molecules is the downregulation of the proto-oncogene C/EBPbeta-LIP that is generated by 

translation re-initiation. Moreover, the small molecules similarly suppress proliferation of breast 

carcinoma cells with concomitant decrease in C/EBPbeta-LIP expression. We anticipate that 

our reporter system will facilitate the search for new therapeutic drugs against cancer and 

other diseases where translationally controlled genes are casually implicated. 

86

CLAUDIA CASANOVA 

87 


High-throughput siRNA screen to identify regulators of VEGF IRES translation 

Peter Sehr 1, Beate Neumann 1, Petra Binninger 2, Claudia Casanova 3, Christian Thoma 3 

1 EMBL Heidelberg, Germany 

2 University Hospital of Freiburg, Germany 

3 University Hospital of Freiburg/ EMBL Heidelberg, Germany 

Hypoxia activates a switch from cap-dependent to IRES-dependent translation that promotes 

tumor angiogenesis and growth (1). Interestingly, the VEGF mRNA can be translated by such 

an IRES mechanism which is stimulated by hypoxia (1). However, the molecular mechanisms 

responsible for VEGF IRES translation are poorly understood. In particular, it has not been 

addressed whether specific factors are necessary for VEGF IRES function. To identify specific 

functional modulators that control, positively or negatively, VEGF IRES-mediated translation 

under hypoxic conditions we performed a high-throughput siRNA screen using a previously 

established in vivo assay based on RNA transfections (2). Our objective is to understand the 

molecular and cellular mechanisms that enable selective translation of VEGF mRNA in cancer 

cells. This could ultimately provide novel in vivo-validated targets for therapeutic intervention. 

We identify seven genes that negatively regulate VEGF IRES translation and two genes that 

positively regulate VEGF IRES function. The hits were identified with at least two independent 

siRNA sequences and validated by using reporter mRNAs for cap-dependent translation as a 

control. The results from the high-throughput siRNA screen and the validation experiments will 

be presented. References (1) Braunstein et al., Mol Cell (2007) (2) Thoma et al., Mol Cell (2004)



REGINA CENCIC 

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, 

Silvestrol 

John A Porco Jr 1, Regina Cencic 2, Marilyn Carrier 2, Gabriela Galicia-Vazquéz 2, Rami 

Sukarieh 2, Jerry Pelletier 2 

1 Boston University, United States of America 


Translation initiation is regulated by eIF4F at the level of the ribosome recruitment step. Levels 

of cellular eIF4F are regulated by the target of rapamycin, mTOR. The extent to which 

translation of specific mRNAs is altered in response to changes in mTOR activity and eIF4F 

levels varies substantially among different transcripts and is largely dependent upon sequence 

elements within each mRNA, such as the presence of discrete hairpin structures in the 5’ 

untranslated regions. The deregulation of the PI3K/Akt/mTOR signaling axis in human cancers, 

the finding that ectopic expression of eIF4E is oncogenic and the demonstration that targeted 

down-regulation of eIF4E displays therapeutic benefit in xenograft models suggest that the 

process of translation initiation is a potential anti-cancer target. We have previously shown that 

the natural product silvestrol can re-sensitize tumor cells to standard-of-care agents in the 

Eμ-myc lymphoma model. Silvestrol inhibits translation initiation by targeting the RNA helicase, 

eukaryotic initiation factor (eIF) 4A subunit of the eIF4F complex, and prevents ribosome 

loading onto mRNA templates. Data will be presented to further elucidate the mode of action 

of silvestrol as an inhibitor of eIF4A, as well as its anticancer activity in human breast and 

prostate cancer xenograft models. 

88

LAURENT CHAVATTE 

Structural elements from the SECIS that determine UGA/selenocysteine 

recoding efficiency 

Donna Driscoll 1, Laurent Chavatte 2, Lynda Latrèche 2, Olivier Jean-Jean 2 

1 Cleveland Clinic, United States of America 

2 CNRS, France 

89 


In humans, the synthesis of twenty-five selenoproteins involves a remarkable mechanism of 

translational recoding of the UGA stop codon into a selenocysteine (Sec), also known as the 

21st amino acid. To do so, the 3'UTR of the selenoprotein mRNAs harbors a Sec Insertion 

Sequence (SECIS). It is composed of two essential motifs: an AAA/G motif in the apical loop 

and a SECIS core containing two sheared tandem GA base pairs responsible for a kink-turn 

folding of the RNA. SBP2 and L30 bind the tandem GA base pair motif of the SECIS, which 

has been suggested to act as a molecular switch characterized by a conformational transition 

upon protein binding. Our data indicate that the nature of the SECIS element acts at the 

translational level to modulate selenoprotein expression, which is highly tuned in mammalian 

cells. To define the precise determinants of the SECIS element, we have used a 

luciferase-based reporter gene system. The firefly luciferase coding region (in which Cys258 is 

mutated to UGA) is linked to various human SECIS elements in an expression vector to assay 

the UGA/Sec recoding activity in transfected cells and in in vitro translation. Using this system, 

we have verified that the selenium status of the culture media modulates the UGA/Sec 

recoding efficiency in cells to the same extent as endogenous selenoproteins. Surprisingly, we 

found that the SECIS elements displayed a wide range of UGA recoding activities, spanning 

several thousand-fold in vivo and several hundred-fold in vitro. We have generated chimeric 

constructs between one weak and one strong element that we cloned downstream of our 

luciferase construct. Within the SECIS elements, we have identified the structural determinants 

responsible for positive and negative regulation of UGA recoding efficiency in cells and in vitro. 

Functional implications of these structural determinants will be analysed and discussed.



CHANGCHUN CHEN 

Defects in tRNA Modification Associate with Neurological and Developmental 

Dysfunctions in Caenorhabditis elegans Elongator Mutants 

Simon Tuck 1, Changchun Chen 2, Anders Byström 2 

1 Umea Centre of Molecular Medicine, Sweden 

2 Umeå University, Sweden 

Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the 

human Elongator homologue, hELP1, are associated with the neurological disease Familial 

Dysautonomia. However, how Elongator functions in metazoans, and how the human 

mutations affect neural functions is incompletely understood. Here we show that in 

Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are 

required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of 

wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. 

elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of 

chemosensory neurons required for salt chemotaxis learning. elpc-1 and elpc-3 gene 

inactivation cause a defect in this process, associated with a posttranscriptional reduction of 

neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and 

elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for 

thiolation of tRNAs having the 5´methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; 

tuc-1 double mutants display developmental defects. Our results suggest that by its effect on 

tRNA modification, Elongator promotes both neural function and development. 

90

WEI WEN CHIEN 

91 


p16INK4a inhibits CDK1 expression in MCF7 cells via the microRNA pathway 

Wei Wen Chien, Régine Catallo 

Université Claude Bernard Lyon 1, CNRS UMR 5239, France 

The p16INK4a protein regulates cell cycle progression mainly by inhibiting the activity of 

cyclin-dependent kinases (CDKs) 4 and 6, the subsequent phosphorylation of retinoblastoma 

protein (pRb) and the release of transcription factor E2F. In addition, p16INK4a represses the 

activity of other transcription factors such as c-myc, NF-κB and c-jun/AP1. Here, we report 

that the ectopic expression of p16INK4a in MCF7 cells provoked a G1 phase cell accumulation 

as well as a lengthening of S phase, suggesting the involvement of downstream cell cycle 

regulatory proteins. The expression of CDK1, an essential cell cycle gene, was down-regulated 

by p16INK4a at the post-transcriptional level. This down-regulation was mediated by the 

3’-untranslated region of CDK1 mRNA and was associated with a modified expression balance 

of microRNAs that potentially regulate CDK1. We provide evidence that p16INK4a 

up-regulated miR-410 and miR-650 expression and that these two miRNAs targeted CDK1. In 

addition, we demonstrate that the induction of miR-410, but not miR-650, was related to the 

inhibitory function of p16INK4a on the pRb/E2F pathway. We thus propose that p16INK4a 

may regulate gene expression at different levels by modifying the functional equilibrium of 

transcription factors and consequently the expression balance of miRNAs.



ANNA CHIRKOVA 

How does 23S rRNA contribute to tRNA movement through the ribosome? 

Anna Chirkova, Matthias Erlacher, Nina Clementi Norbert Polacek 

Innsbruck Medical University, Biocenter, Austria 

Years of ribosome explorations made it clear that the main role in the catalytic activity of the 

ribosome is provided by the ribosomal RNA. Chemical engineering and site-directed 

mutagenesis of the 23S rRNA are combined here together to precisely study the function of 

selected residues in the peptidyl transferase center (PTC). The principle and the main 

advantage of this “atomic mutagenesis” approach (so called gapped-cp-reconstitution of 50S 

ribosomal subunits) consist in the precise incorporation of diverse non-natural nucleoside 

analogues at 23S rRNA sites of interest. 

Comparison of the atomic mutagenesis effects on the in vitro translation system with the 

effects obtained in single turnover reactions of the elongation cycle gives us additional 

information about the functions of PTC nucleosides. Removal of single nucleobases from the 

PTC nucleosides predicted to be involved in peptide bond formation or tRNA translocation 

(A2602, U2585 or A2451), did not significantly affect polyU-directed synthesis of (Phe)n 

peptides. However, ribosome mutations causing disruption of universally conserved pair 

C2063-A2450, strongly decreased product formation in the polyU-directed in vitro translation 

system, while having little effect on peptide bond formation as well as on EF-G binding and 

GTPase activation. Further analysis of the (Phe)n chain lengths revealed that production of 

peptides longer than (Phe)2 is impaired on the ribosomes with the broken C2063-A2450 pair. 

This evidence indicates that the integrity of the C2063-A2450 base pair in the PTC is essential 

for effective tRNA translocation during protein synthesis. 

92

BETTY CHUNG 

93 


A small slip back for the ribosome reveals more than just moving forward 

Betty Chung, Andrew Firth, John Atkins 

Recoding Lab, BioSciences Institute, University College Cork, Ireland 

Programmed ribosomal frameshifting is a mechanism that allows the synthesis of alternative, 

N-terminally coincident, C-terminally distinct, proteins from the same RNA. Many viruses utilize 

frameshifting to optimize the coding potential of compact genomes, circumvent the host cell’s 

canonical rule of one functional protein per mRNA, or to express alternative proteins in a fixed 

ratio. Programmed frameshifting is also used in a small number of cellular genes. Recently we 

reported a new example of ribosomal -1 frameshifting. It occurs with an estimated efficiency of 

~5 - 50%, depending on species, at a conserved UUUUUUA motif within the sequence 

encoding the alphavirus 6K protein. The frameshifting results in the synthesis of an additional 

small protein, termed ‘TF’ (TransFrame protein, ~8 kDa) [1]. This new case of frameshifting was 

unusual in that the -1 frame ORF was very short, and completely embedded within the 

sequence encoding the overlapping polyprotein. 

Here, we report an analysis of the remarkable diversity among the 3’signals predicted to 

stimulate efficient frameshifting at the UUUUUUA motif. While many alphavirus species utilizes 

a 3’ RNA structure such as a hairpin or pseudoknot, some species apparently lack any 3’ 

stimulatory structure. Instead, up to 10% frameshifting can be achieved through stimulation by 

just the 20 nucleotides 3’-adjacent to the shift site. In addition, the identity of the shift site itself 

is important for efficient frameshifting within the context of Semliki Forest virus. These 

experimental and bioinformatics analyses have expanded the repertoire of -1 frameshifting 

stimulators in mammalian systems. 

[1] Firth, A., Chung, B., Fleeton, M., & Atkins, J. (2008). Discovery of frameshifting in alphavirus 

6k resolves a 20-year enigma. Virol J, 5(1), 108.



BRYAN CLARKSON 

Examining Functions of eIF4G Isoforms in Saccharomyces cerevisiae 

Bryan Clarkson, Wendy Gilbert, Jennifer Doudna 

University of California, Berkeley, United States of America 

Eukaryotic initiation factor 4G (eIF4G) plays a central role in translation, serving as a scaffold to 

nucleate a number of molecules whose positioning is critical for efficient initiation. Most 

eukaryotic genomes encode multiple eIF4G gene variants. Although previous work has 

revealed functional differences between eIF4G isoforms, their specific roles in translation 

remain a mystery. Using genomic, genetic, and biochemical approaches, we are exploring the 

function of two eIF4G variants, encoded by TIF4631 and TIF4632, in Saccharomyces 

cerevisiae. 

Under standard laboratory conditions deletion of TIF4631 causes a growth defect and 

decreased levels of translation initiation, whereas TIF4632Δ cells exhibit wild-type growth and 

translation profiles. Yeast express three times more Tif4631 than Tif4632. Therefore, the 

TIF4631Δ-associated phenotypes may result from the removal of a larger fraction of total 

eIF4G and not the absence of an isoform-specific functionality. 

To investigate this, we constructed “homogenic” strains, in which the open reading frame of a 

single isoform was placed at both eIF4G loci, resulting in cells with similar overall levels, but 

distinct types, of eIF4G. Normalization of eIF4G amounts restored growth and translation 

initiation rates to wild type levels. Higher resolution analysis using polysome microarrays 

revealed that no mRNAs rely exclusively on a specific eIF4G isoform for their translation. 

Therefore, under standard laboratory conditions, there exists a large functional overlap 

between eIF4G isoforms. 

To further explore eIF4G functionality, we are systematically investigating epistatic interactions 

between each isoform and other yeast genes and examining the fitness of the homogeneic 

strains under a variety of environmental conditions. These experiments will further define the 

roles of eIF4G variants and have the potential to identify novel mechanisms of translational 

control. 

94

LAURA COBBOLD 

95 


A mutant form of the c-myc IRES has increased interactions with the ITAFs 

PTB and YB-1 

Anne Willis, Laura Cobbold, Lindsay Wilson, Alexandra Kondrashov, Keith Spriggs, Martin 

Bushell 

Nottingham University, United Kingdom 

It has been shown previously that there is 10- to 25-fold increase in c-Myc protein expression 

in Mutiple Myeloma (MM) both in material directly obtained from patients and in derived cell 

lines. This increase is not accompanied by a corresponding increase in the overall level of 

c-myc mRNA suggesting that de-regulated expression of c-Myc occurs at the level of 

translation. In agreement with these data a mutant version of the c-myc IRES, containing a 

single C-T nucleotide change is present in 42% of cells obtained from MM patients and this 

IRES has greater activity in vivo than the wildtype IRES. Recently, we have identified the 

proteins YB-1, PTB-associated splicing factor (PSF), p54nrb, GRSF1 and PTB as c-myc IRES 

trans-acting factors. The interaction of these proteins with the wild-type and mutant versions 

of the c-myc IRES have been investigated and we show that YB-1 and PTB both interact with 

the region of the mutant IRES containing the nucleotide base change, and bind to the mutant 

version x and y fold more tightly. Moreover these protein and stimulatory the activity to the 

mutant form of the IRES x times than the wildtype IRES. Interestingly, these proteins are 

shown to form a native complex both in vivo and in vitro. Finally we show the levels of these 

two ITAFs are altered in MM derived cell lines. These data suggest that the deregulated 

expression of c-Myc in MM results from enhanced IRES activity due to increased ITAF binding 

to a mutant form of the c-myc IRES.



BRITTA COORDES 

Translational regulation by phosphorylation of ribosomal proteins 

Katja Straesser 1, Britta Coordes 2, Katharina Brünger 2, Ivan Matic 3, Jesper Olsen 3, Matthias 

Mann 3 

1 Gene Center, LMU Munich, Germany 

2 LMU Munich, Germany 

3 MPI Biochemistry Martinsried, Germany 

Translational regulation has been extensively studied and was found to be mainly mediated at 

the step of initiation. There, the phosphorylation of initiation factors is the most prominent 

means of regulation. In contrast, little is known about the regulation of translation by the 

phosphorylation of ribosomal proteins. 

Our lab identified the cyclin dependent kinase Ctk1 as a novel regulator in translation. Ctk1 is 

the kinase component of the C-terminal-domain kinase I (CTDK-I) complex, which is important 

for transcription elongation by phosphorylating the C-terminal-domain (CTD) of RNA 

polymerase II (PolII). Interestingly, our group showed that Ctk1 also functions in translation by 

phosphorylating the ribosomal protein Rps2, thereby increasing translational fidelity during 

elongation. Recently, we discovered an additional role of Ctk1 in translation initiation. In the 

absence of Ctk1 the formation of 80S ribosomes during translation initiation is impaired, while 

48S initiation complexes accumulate. 

As a global approach we aim to identify new, biologically relevant phosphorylation sites on 

ribosomal proteins. We applied phosphoproteomic methods to analyse the phosphorylation 

status of different ribosomal populations yielding a comprehensive catalogue of ribosomal 

phosphorylation sites in S. cerevisiae. In order to identify biologically important phosphosites in 

stress conditions, we investigated changes in the phosphorylation pattern of ribosomal 

proteins in response to stress using Stable Isotope Labelling by Amino Acids (SILAC). 

The investigation of the biological function of the newly identified phosphorylation sites by 

biochemical means as well as the elucidation of the molecular function of Ctk1 in translation 

initiation might provide new aspects of translational control. 

96

LUCIE CUCHALOVA 

97 


Yeast eIF3g promotes resumption of scanning of post-termination ribosomes 

as a part of the GCN4 reinitiation mechanism whereas eIF3i stimulates 

processivity of scanning 

Lucie Cuchalova, Leos Valasek, Tomas Kouba 

Academy of Sciences of the Czech Republic, Institute of Microbiology, Czech Republic 

Eukaryotic translation initiation factor 3 (eIF3) is the most complex yet, on the molecular level, 

the least understood factor implicated in stimulation of virtually all initiation steps including 

recruitment of Met-tRNAiMet and mRNA to the 40S ribosome. Here we describe functional 

characterization of two small essential core subunits eIF3i and eIF3g from Saccharomyces 

cerevisiae thought to be dispensable for the latter key eIF3 roles both in yeast and mammals. 

Specific mutations targeting the RRM of yeIF3g or the WD40 repeat #6 of yeIF3i both impair 

the induction of GCN4 translation that occurs via reinitiation. However, whereas the yeIF3i 

mutant prevents full GCN4 derepression by decreasing the rate of scanning and increasing the 

rate of leaky scanning, the yeIF3g-RRM mutant impedes 40S ribosomes terminating at the first 

uORF1 of the GCN4 leader to resume scanning for reinitiation downstream. Remarkably, 

detailed genetic analysis and localization of the yeIF3g position on the 40S ribosomes revealed 

that the yeIF3g-RRM promotes resumption of scanning by a distinct mechanism than that that 

was recently described for the N-terminal domain of yeIF3a. Together, our results provide the 

first in vivo evidence that both yeIF3g and eIF3i subunits stimulate translational steps following 

formation of the 48S pre-initiation complex. The study was supported by HHMI and the 

Wellcome Trust.



JOSEPH CURRAN 

Impact of alternative 5'UTRs on the translational expression of the human 

mdm2 (hdm2) and elk-1 mRNAs 

Joseph Curran, Tanguy Araud, Raphael Genolet, Pascale Jaquier-Gubler 

University of Geneva Medical School, Switzerland 

The lab is interested in understanding how changes in the organisation of the 5’ UTR impacts 

on the translation read-out, and how such changes are exploited in the regulation of cell 

growth and differentiation. Work focuses on two cellular oncogenes, elk-1 and hdm2. The 

former is a transcription factor that plays a key role in the IEG response during proliferation. We 

have characterised two transcripts that arise as a result of alternative splicing within the 5’ 

UTR. Both long and short contain features that play a role in ribosomal access to the ELK-1 

AUG including structure and uORFs. The second uORF contains two codons and directs 

re-initiation at downstream start sites. The rat elk-1 mRNA expresses a second protein 

(sELK-1) by de-novo initiation on the seventh AUG. This protein is expressed in rat neuronal 

cells and in PC12 cells during NGF driven differentiation. However, we have been unable to 

mimic this translational switch using a human cDNA. Recent results have shown that the 

alternatively spliced exonII in humans and rat is different opening the possibility that changes in 

the organisation of the 5’UTR may be modulating access to the sELK-1 AUG. This is currently 

being tested. HDM2 is an E3 ubiquitin ligase that serves to regulate the intracellular levels of 

the P53 tumour suppressor. Its mRNA also has two 5’ UTRs arising by the use of alternative 

promoters. The long comes from the constitutive P1 promoter whereas the short is expressed 

from the P53 activated P2 promoter. The hdm-2 mRNA was fished-out of a high-throughput 

translational profiling screen whose aim was to identify transcripts exhibiting rapamycin 

resistance. Results indicate that neither 5’ UTR possesses IRES activity although both confer 

rapamycin resistance to a reporter construct. Our recent work confirms that this phenomenon 

is coupled to mTOR signalling. 

98

ANDREAS CZECH 

99 


Influence of stress conditions on tRNA composition and translation efficiency 

Andreas Czech, Zoya Ignatova 

University Potsdam, Germany 

Protein pattern differs in cells comprising various organs and at different developmental stage 

(mitotic and postmitotic) and concentrations of tRNA species are directly proportional to the 

amino acid demand and composition of proteins in these conditions. In addition, cells rapidly 

reorganize their protein expression pattern in response to stress stimuli, e.g., oxidative or 

osmotic stress. Thereby the mRNA level remains mostly unaltered suggesting a control point at 

the translational level. Modulating tRNA levels is a key to regulate protein synthesis at the level 

of translation elongation; proteins up-regulated under stress may include slow-translating 

codons that limit protein abundance in normal conditions. A stress-regulated increase of the 

concentration of some tRNAs will accelerate the translation and increase the abundance of 

stress-related proteins. Currently we are determining variations in the tRNAs concentration in 

mammalian neuroblastoma cells (murine N2a, human SH-SY5Y cells) under osmotic and 

oxidative stress. The rate of translation of each codon is dependent on the recharging of the 

cognate tRNA and on the delivery of the ternary complex to the ribosome driven by passive 

diffusion, which in turn is strongly proportional to the tRNA concentration. Determining the 

accurate tRNA concentration in normal growth und under stress conditions will enable us to 

accurately determine translational kinetics of the proteome under these conditions. 

Additionally, it will shed light on the translational regulation of protein expression under stress 

conditions.



EDWARD DARZYNKIEWICZ 

Enzymatically Stable and Translationally Highly Effective Phoshorothioate Cap 

Analogs 

Warsaw University, Poland 

E. Darzynkiewicz 1 , J. Kowalska, J. Jemielity 1 , J. Zuberek 1 , M. Lukaszewicz 1 , 

E. Grudzien-Noglaska, M. Lewdorowicz, E. Bojarska, M. Ziemniak, J. Stepinski, R.E. Rhoads 2 , 

R.E. Davis 3 

1 University of Warsaw, Poland 

2 Louisiana State University, USA 

3 University of Colorado, USA 

The synthesis and properties of dinucleotide phoshorothioate cap analogs will be presented. 

This serie covers six dinucleotide cap analogs bearing a single phosphorothioate modification 

at either the α, β, or γ position of the 5’,5’-triphosphate chain. Three of them were also 

modified with methyl groups at the 2’-O position of 7-methylguanosine to produce 

anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the 

phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 

and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. It has 

been found that phosphorothioate modifications generally stabilized the complex between 

eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer 

of the β-substituted analog m7GppSpG) was characterized by a KAS that was more than 

4-fold higher than unmodified m7GpppG. Analogs modified in the γ position were resistant to 

hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and C. 

elegans sources. The analogs resistant to DcpS act as potent inhibitors of in vitro protein 

synthesis in rabbit reticulocyte lysates. Luciferase mRNA capped with m27,2’-OGppSpG (D2) 

was translated 5-fold more efficiently in HC11 cells than its counterpart with m7GpppG. In 

summary, the newly synthesized phosphothioate cap analogs, incorporated into mRNAs, can 

be used as highly effective “translators”. 

100

MARIE-CLAIRE DAUGERON 

The yeast GTPases Rbg1 and Rbg2 are implicated in translation 

101 


Manoel Prouteau 1, François Lacroute 1, Bertrand Séraphin 1, Marie-Claire Daugeron 2 


2 Equipe labellisée La Ligue, CGM – CNRS, avenue de la Terrasse, 91198 Gif sur Yvette 

Cedex, France and Université Paris Sud XI, France 

Rbg1 and Rbg2 from Saccharomyces cerevisiae belong to a highly conserved subfamily of 

GTP binding proteins found in a wide variety of archaeal and eukaryotic species. The exact 

function of these factors is still unknown. We present here a set of data strongly indicating that 

Rbg factors play a role in translation. We first have identified Rbg1 and Rbg2 associated 

proteins by TAP purification and mass spectrometry. These data demonstrate that Rbg1 and 

Rbg2 are part of two distinct protein complexes. Accurate measurement of synthetic 

interactions at the genomic scale (Decourty et al., Proc Natl Acad Sci USA. (2008) 105, 

5821-5826) revealed a functional redundancy of the Rbg's protein complexes. Thus, we 

performed a genetic screen for synthetic slow growth or lethal mutants with a Δrbg1Δrbg2 

strain. This identified a novel factor functionally related to the Rbg complexes. Interestingly, 

mutation of this factor produced severe growth defects and altered polysome profiles when 

associated with various combinations of mutants of the various subunits of the Rbg 

complexes. We also observed that Rbg1, Rbg2 and their partners are present in polysomes 

after fractionation in sucrose gradient. Finally, we also identified physical and genetic 

interactions between Rbg's and known regulators of translation. Altogether, these data 

indicate that the Rbg1 and Rbg2 function is linked to translation. This suggests that more 

GTPases are implicated in eukaryotic translation than assumed by current models.



RICHA DAVE 

Direct observations of the tetrameric ribosomal stalk protein L12 in the 

multistep process of tRNA selection 

Richa Dave, Roger Altman, Peter Geggier, Daniel Terry, Scott Blanchard 

Weill Cornell Medical College, United States of America 

The conserved GTPases Elongation Factor-Tu (EF-Tu) and EF-G bind non-competitively to the 

ribosomal A site via interactions with the highly mobile, large (50S) ribosomal subunit protein 

L12. Through direct interactions with L12, as well as the neighboring GTPase activating center 

(GAC) of the 50S subunit, EF-Tu and EF-G increase the rate and fidelity of tRNA selection and 

translocation, respectively. Mutational studies as well as depletion of L12 from the ribosome 

suggest that L12 plays a direct role in factor recruitment, GTP hydrolysis and inorganic 

phosphate release. Detailed structural information regarding L12’s role during elongation 

factor-mediated processes has thus far been hampered by its absence from high-resolution 

structures of the ribosome. Here we report direct observations of L12’s participation in the 

multistep process of tRNA selection through the use of high- spatial and –time resolution 

single-molecule fluorescence resonance energy transfer (smFRET) imaging. In order to directly 

monitor time-dependent, nanometer-scale conformational changes of the L12 protein during 

the process of tRNA selection, ribosome complexes were site-specifically labeled on the 

C-terminal domain (CTD) of L12, where FRET could be detected with respect to components 

of the ternary complex (EF-Tu and aa-tRNA) as well as the GAC (L11). Using this system we 

show that a single CTD of L12 assists in the initial binding of the ternary complex to the 

ribosome, directly facilitating EF-Tu’s interaction with GAC to stabilize the state where GTP 

hydrolysis can occur. These data suggests a model in which following EF-Tu-catalyzed GTP 

hydrolysis, the L12 CTD dissociates from EF-Tu to precipitate inorganic phosphate release and 

conformational changes in EF-Tu that allow aa-tRNA accommodation into the A-site. 

102

ALEXANDRE DAVID 

103 


Aminoacyl synthetases Reveal Compartmentalization of Protein Translation 

Alexandre David 1, Michael Brad Strader 1, Nir Netzer 2, Suman Das 2, Jonathan W. Yewdell 2, 

Jack R. Bennink 2, Jeffrey Goodenbour 3, Tao Pan 3 

1 NIH, United States of America 

2 NIH / NIAID, United States of America 

3 University of Chicago, United States of America 

Transfer RNAs (tRNAs) are information adaptor molecules, decoding RNA information into 

amino-acid sequence. The faithful translation of mRNA into protein requires highly accurate 

coupling of amino acids to their cognate tRNAs. This is accomplished by twenty different 

tRNA aminoacyl synthetases (aaRSs); one for each of the common amino acids. In 

collaboration with Tao Pan and colleagues, we recently discovered that the fidelity of 

attachment of Met to its cognate tRNAs is altered by infectious and chemical stressors, and 

that this misacylation leads to Met substitutions in proteins. To elucidate the mechanism 

behind this remarkable stress-mediated Met-misacylation, we focused our attention on the 

cellular biology of the aaRSs. We were particularly interested in the nine synthetases that 

comprise the multi-aaRS complex (MSC), which is directly implicated in the Met misacylation 

phenomenon. Using a novel immunofluorescence-based method to identify actively translating 

ribosomes, we found that the MSC associates with translating ribosomes and dissociates 

rapidly upon blocking translation via multiple modalities. Parallel findings were made by 

biochemical characterization of ribosomes and MSC following cellular fractionation. The close 

association of translating ribosomes and MSC was clear following infection of cells with either a 

poxvirus or an alphavirus, where the MSC localized to focal zones of intense translation of viral 

gene products. These findings demonstrate that translation is a highly organized process that 

entails the formation of translation compartments consisting of translating ribosomes and 

aaRSs. Moreover, we show that some viruses exploit this compartmentalization to selectively 

shut down host translation and enhance the synthesis of their own proteins. Finally, we provide 

a foundation for understanding the modulation of aaRS fidelity under cell stress conditions.



LUIGI DE COLIBUS 

Structural studies of Nhm1, a key enzyme in the nuclear and cytoplasmic 

metabolism of RNA 

Lijie Sun 1, John E.G. McCarthy 1, Luigi De Colibus 2, Robert Gilbert 2 

1 Interdisciplinary Biocentre, University of Manchester, United Kingdom 

2 Oxford University, United Kingdom 

mRNA turnover plays a key role in control of gene expression both by setting the basal level of 

gene expression and as a site of regulatory responses. Two general paths of mRNA decay 

have been identified downstream of the deadenylation event in eukaryotic cells. -a decapping 

enzyme consisting of two subunits, Dcp1p and Dcp2p, removes the 5' cap structure, exposing 

the transcript to digestion by a 5'→ 3' exonuclease, and generating m7GDP. -mRNAs can be 

degraded in a 3'→5' direction by the cytoplasmic exosome. This generates a range of 

m7Gppp-oligonucleotides. While heterodimeric Dcp1-Dcp2 decaps full-length mRNAs, the 

DcpS type of “scavenging” enzyme typically hydrolyses the m7GDP and m7Gppp- 

oligonucleotides generated by the two pathways above. Recently, we have identified and 

biochemically characterized a Schizosaccharomyces pombe DcpS ortholog, called Nhm1. 

Unlike mammalian DcpS and yeast Dcs1, this enzyme is able to catalyse the decapping of 

capped mRNAs considerably longer than 10 nucleotides. Fluorescence and 

immunofluorescence microscopy show that Nhm1 is located both in the nucleus and in the 

cytoplasm. We have solved crystal structures for Nhm1 in the Apo form, in complex with a 

non-hydrolysable GTP analogue, and in complex with m7GpppG as an H244N mutant. This 

mutation prevents enzyme turnover and has allowed us to view atomic details of Nhm1’s 

characteristic catalytic histidine triad motif, and shed light on the catalytic mechanism of the 

enzyme. Catalysis involves the generation of an asymmetric dimer in which one monomer is in 

an open conformation and inactive and the other in a closed conformation and active. Each 

monomer alternatively swaps between conformers via a 30 Å swing. Altogether these data 

provide new insight into the mechanism of action of an enzyme that is involved in both 

cytoplasmic and nuclear RNA metabolism. 

104

OSVALDO DE MELO NETO 

105 


Two eIF4G Homologues from Trypanosomatids Display Functional Properties 

Compatible With Roles In Two Diverged eIF4F Complexes 

Osvaldo de Melo Neto, Danielle M. N. Moura, Christian R. S. Reis, Rodrigo P. Lima, Regina 

C. B. Q. Figueiredo 

Centro de Pesquisas Aggeu Magalhães / Fundação Oswaldo Cruz, Brazil 

The trypanosomatids are pathogenic protozoan which include species belonging to the genera 

Trypanosoma and Leishmania and are characterized by unique molecular mechanisms 

associated with their gene expression. Translation initiation is not well known but may include 

potentially novel processes not yet observed in other organisms. In higher eukaryotes, the 

scaffolding protein eIF4G, part of the eIF4F complex, performs many functions in translation 

initiation related to ribosome recruitment to the mRNA. Here we describe work aimed at the 

functional characterization of the trypanosomatid eIF4G homologues, five of which have been 

described (LmEIF4G1-5 in L. major). Through pull-down assays, two of these (LmEIF4G3-4) 

were found to bind to Leishmania homologues of the remaining eIF4F subunits, eIF4A and 

eIF4E, and were selected for further studies. The two proteins differ in the binding to eIF4A and 

bind to different eIF4E homologues. The eIF4E binding sequences were located to their short 

N-terminal regions, but site directed mutagenesis of selected residues uncovered binding 

motifs which differ for each protein and which are also distinct from the consensus described 

from other eukaryotes. To study these proteins in vivo, we opted to use T. brucei and selected 

their orthologues (TbEIF4G3-4), plus TbEIF4G5, for further analysis. The three proteins were 

found to be cytoplasmic and all were essential for cellular viability after knock-down through 

RNAi. Depletion of TbEIF4G3 led to a quick reduction in translation with subsequent cellular 

death whilst depletion of TbEIF4G4-5 produced a more delayed growth retardation effect prior 

to cell death. Cell lacking TbEIF4G4 also exhibited changes in morphology but no substantial 

inhibition in protein synthesis. In all our results are consistent with the existence of at least two 

distinct eIF4F complexes, with the one containing TbEIF4G3 having a major role in translation.



CORNELIA DE MOOR 

The polyadenylation inhibitor cordycepin disrupts mTOR signalling 

Cornelia de Moor 1, Ying Ying Wong 1, Alice Moon 1, Ruth Duffin 1, Adeline Barthet-Barateig 1, 

Hedda Meijer 1, Michael Clemens 2 

1 University of Nottingham, United Kingdom 

2 University of Sussex, United Kingdom 

Cordycepin (3’ deoxyadenosine) is found in the parasitic fungus Cordyceps miltaris and has 

been proposed as the active component of traditional medication that is reputed to alleviate a 

large variety of ailments. It is a known polyadenylation inhibitor with a large spectrum of 

biological activities, including anti-proliferative, pro-apoptotic and anti-inflammatory effects. In 

this study, we confirm that cordycepin causes a decrease in the poly(A) tail size of specific 

mRNAs with some mRNAs being much more sensitive to cordycepin than others. Low doses 

of cordycepin cause a decrease in cell proliferation. At high doses, however, cordycepin 

prominently affects cell adhesion and indirectly reduces protein synthesis to very low levels. It 

shuts down a signal transduction pathway, the mTOR pathway, which is known to control 

proliferation, cell adhesion and protein synthesis. In contrast to rapamycin, cordycepin inhibits 

the activities of both the mTORC1 and the mTORC2 complexes, affecting the activity of the 

protein kinase Akt. Adenosine is a cordycepin antagonist and inhibitors of adenosine import 

and phosphorylation prevent the effect of cordycepin on protein synthesis, indicating that this 

drug is acting intracellularly and needs to be converted to cordycepin monophospate. 

Cordycepin was also shown to function as an activator of the AMPK pathway. An inhibitor of 

AMPK blocked cordycepin mediated inhibition of translation and Akt dephosphorylation, 

indicating the effects of cordycepin on translation and mTOR signalling are mediated by its 

activation of AMPK. These effects of cordycepin explain most of the observations reported in 

tissue culture experiments and provide a mechanistic explanation for the action of this agent as 

an anti-proliferative and anti-inflammatory drug. 

106

SILVIA DE RUBEIS 

107 


CYFIP1, a neuronal eIF4E-BP, links local translational regulation to spine 

remodeling: insights into the Fragile X Syndrome 

Claudia Bagni 1, Silvia De Rubeis 2, Ka Wan Li 3, August B. Smit 3 

1 Center for Hum Genet, KU Leuven, Belgium; Dept Mol and Dev Genet, VIB, Leuven, 

Belgium; Dept Exp Medicine Biochem Sci, Univ Tor Vergata, Roma, Italy 

2 Dept Biology, Univ "Tor Vergata", Roma, Italy; 2Center Hum Genet, KU Leuven, Belgium; 

Dept of Mol and Dev Genet, VIB, Leuven, Belgium 

3 Dept of Mol and Cell Neurobiology, Center for Neurogenomics and Cognitive Research, VU 

University Amsterdam, Amsterdam, The Netherlands 

Fine regulation of mRNA transport and translation at synapses underlies synaptic plasticity and 

brain development. One of the key molecules implicated in this process is the Fragile X Mental 

Retardation Protein (FMRP), the protein lost in the mental retardation form called Fragile X 

Syndrome (FXS). We have recently demonstrated that FMRP represses translation initiation via 

its cytoplasmic interacting protein CYFIP1/Sra1, known as a regulator of the actin 

cytoskeleton. FMRP tethers a specific subset of neuronal mRNAs to CYFIP1, which can in turn 

bind the translation initiation factor eIF4E, and thus blocks the access of eIF4G. After neuronal 

stimulation, the CYFIP1-eIF4E complex is released and protein synthesis ensues. 

By using CYFIP1 immunoprecipitation from different subcellular compartments of the neuron 

and protein identification by mass spectrometry, we found new interactors of the 

CYFIP1-FMRP particle assembled in specific molecular complexes according to their 

subcellular location. Some of these factors are specifically involved in mRNP transport, others 

in translational regulation and a third class seems to be mainly involved in cytoskeleton 

remodeling and anchoring to membrane receptors. In particular, we were able to (a) identify 

new molecular partners of CYFIP1 that link protein synthesis to the cytoskeleton, (b) 

demonstrate that actin-remodelling factors have an effect on the CYFIP1-FMRP-eIF4E 

complex, and (c) show that the translational machinery physically associates with membrane 

receptors and scaffolding proteins. 

These findings provide novel evidence for the interplay of local translational regulation and 

cytoskeleton remodeling. Neuronal activity through receptor activation would control this 

process, leading to synaptic macromolecular changes and enabling synaptic plasticity.



SEBASTIAN DE VRIES 

The role of 3’UTR binding factors in VEGF IRES mediated mRNA translation 

Sebastian de Vries 1, Susan Weinlich 1, Nadine Flach 1, Dirk H. Ostareck 2, Antje 

Ostareck-Lederer 2 

1 Martin-Luther-University Halle-Wittenberg, Germany 

2 University Hospital, RWTH Aachen, Germany 

The vascular endothelial growth factor (VEGF) is one of the most important players in both 

physiological and pathological angiogenesis. Many cancer types (e.g. breast cancer) were 

shown to be associated with high VEGF expression. Low oxygen levels (hypoxia) in growing 

solid tumors lead to an increase of VEGF expression resulting in tumor vascularisation. Insight 

in the molecular mechanisms, which control VEGF expression might help to develop tools for 

alternative VEGF-targeting cancer therapies. While the expression of VEGF is well studied on 

the transcriptional level, there are still open questions concerning its post-transcriptional 

control. Translation initiation of VEGF mRNA is mediated 5’cap independent by two internal 

ribosomal entry sites (IRES) A and B. Regulatory functions of the VEGF mRNA 3’UTR in 

translation control are not investigated in detail. 

Our work focuses on the identification and functional characterisation of cellular factors that are 

involved in regulation of VEGF IRES driven translation and the role of the VEGF mRNA 3’UTR. 

As VEGF levels are increased in breast cancer tissues we developed an in vitro translation 

system based on cytoplasmic extract of the human breast cancer cell line MCF-7. This extract 

is a valuable tool that recapitulates 5’cap-dependent translation, as well as 5’cap-independent 

translation of VEGF 5’UTR bearing reporter mRNAs. 

To isolate potential regulating factors we employ RNA affinity purification approaches. 

Candidates identified by mass spectrometry will be validated in vivo and in vitro. 

108

SILVERA DEBORAH 

Translational regulation of the epithelia to mesenchymal transition in 

inflammatory breast cancer 

109 


Ladan Zolfaghari 1, Silvera Deborah 2, Robert Schneider 2, Rezina Arju 3, Farbod Darvishian 3, 

Judith Goldberg 3, Tsivia Hochman 3, Silvia C Formenti 3, Paul H Levine 1 

1 The George Washington University School of Public Health and Health Services, United 


2 NYU School of Medicine, United States of America 

3 NYU School of Medicine and Cancer Institute, United States of America 

Inflammatory breast cancer (IBC), the most lethal form of breast cancer, presents as an 

unusual disease that generates highly metastatic cell clusters (emboli). A hallmark of IBC is 

overexpression of E-cadherin, which is localized at the cell surface in the adherens junction (AJ) 

complex with p120- and β-catenins. Loss of E-cadherin, a marker for the epithelial to 

mesenchymal transition (EMT) can be induced by a variety of signaling pathways including Wnt 

signaling through β-catenin. E-cadherin promotes IBC metastasis by supporting strong 

interactions within the tumor emboli, leading to passive metastasis of the emboli, or an active 

process termed collective invasion. 

We previously reported that many of the pathological disease characteristics of IBC, such as 

E-cadherin overexpression, result in large part from the overexpression of eIF4GI and its ability 

to drive a high level of translation of IRES-containing mRNAs including that of p120 (Nature Cell 

Bio, 2009). Silencing eIF4GI by shRNA in IBC cells slightly reduced global protein synthesis, 

but selectively inhibited IBC tumor growth and angiogenesis in animal models. eIF4GI silencing 

selectively decreased translation from IRES-containing p120 mRNAs, leading to destabilization 

of E-cadherin, dissolution of tumor emboli and reduction of invasiveness. eIF4GI silencing 

promoted an increase in the levels of several markers for EMT, likely through signaling by 

unanchored β-catenin, which is released into the cytoplasm after AJ dissolution and partially 

relocalized to the nucleus. Thus, we show that availability of a major transcription factor 

(β-catenin) required for metastasis of breast cancer cells is controlled through eIF4GI 

IRES-dependent translation of p120. Results describing the eIF4GI dependent EMT in IBC as 

well as its possible role in modulating EMT in other breast cancer cell lines will be presented.



JOANA DESTERRO 

Auto-regulation of the heterodimeric splicing factor U2AF 

Angela Kramer 1, Nicolas Antih 1, Hansruedi Mathys 2, Witold Filipowicz 2, Joana Desterro 3, 

Teresa R Pacheco 3, Maria Carmo-Fonseca 3 

1 Department Cell Biology, University of Geneve, Switzerland 

2 Friedrich Miescher Institute, Switzerland 

3 Institute of Molecular Medicine, Lisbon, Portugal 

The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, 

a large, 65-kDa subunit (U2AF65) and a small subunit, U2AF35. Our lab has recently shown 

that specific U2AF65-targeting siRNA caused a significant knockdown of both U2AF65 and 

U2AF35 protein levels. This result reveals a feedback mechanism by which depletion of the 

U2AF large subunit triggers the down-regulation of the small subunit. Such a feedback loop 

can be consistent with regulation of U2AF35 by U2AF65 at either transcription or 

post-transcriptional levels. An expected role of U2AF65 on transcription and/or processing of 

U2AF35 mRNA were initially postulated however quantitative RT-PCR results show that 

U2AF65 down-regulation does not affect U2AF35 mRNA levels. We have been focused on 

trying to understand the mechanism underlying this feed-back loop modulated by U2AF65 and 

the results suggest that U2AF65 can bind to the 3ÚTR of U2AF35 mRNA and act as a positive 

modulator for its expression. U2AF65 shuttles between the nucleus and cytoplasm and these 

data is unveiling a new role for this splicing factor in controlling gene expression independently 

of its well known role in splicing. 

110

THOMAS DEVER 

111 


Requirement for Kinase-induced Conformational Change in eIF2a Restricts 

Phosphorylation of Ser51 

Thomas Dever 1, Madhusudan Dey 1, Frank Sicheri 2 

1 NIH, United States of America 

2 Samuel Lunenfeld Research Institute - University of Toronto, Canada 

Phosphorylation of eIF2α on Ser51 is a conserved mechanism to down-regulate cellular protein 

synthesis. Docking of the OB-fold domain of eIF2α, principally around the residue Asp83, on 

helix αG in the PKR C-terminal lobe positions Ser51 in the vicinity of the kinase active site. 

Comparison of the structure of free eIF2α versus eIF2α in complex with PKR revealed that 

Ser51 must be re-positioned ≈20 angstroms to access the phospho-transfer site. We set up a 

genetic screen to identify eIF2α mutations that bypass the kinase-substrate recognition 

mediated by the helix αG-OB fold interaction. The PKR helix αG mutant T487A is non-toxic in 

yeast due to poor eIF2α phosphorylation. An L47I mutation in eIF2α restored PKR-T487A 

toxicity in yeast. Interestingly, the residue Leu47 is one component of a hydrophobic network 

(Leu47, Leu50, Ile58, Ile62) that restricts the position of Ser51 in free eIF2α. Mutations that 

alter residues of the hydrophobic network enable phosphorylation of Ser51 by PKR helix αG 

mutants and by the unrelated kinase PKCα. 

Based on the structure of the PKR-eIF2α complex, we proposed that the docking interaction 

between eIF2α strand β5 and PKR helix αG induces a conformational change that is 

transmitted to the hydrophobic network via the linking residue Ser85. Consistently, while 

mutation of Asp83 in eIF2α, like mutation of helix αG in PKR, impaired Ser51 phosphorylation, 

mutation of the hydrophobic network or of Ser85, which links the Asp83-helix αG contact with 

the Ser51 region of eIF2α, restored phosphorylation. We propose that the protected state of 

Ser51 in free eIF2α prevents phosphorylation by heterologous kinases and that docking of 

eIF2α on PKR helix αG induces a conformational change that exposes Ser51 and thus restricts 

phosphorylation to the proper kinases.



ELENA DOBRIKOVA 

Association of herpes simplex virus (HSV-1) proteins ICP27 and UL47 with 

polyA-binding protein (PABP) 

Elena Dobrikova, Matthias Gromeier, Mayya Shveygert 

Duke University Medical Center, United States of America 

All human pathogenic viruses manipulate the cellular translational machinery to ensure efficient 

translation of viral genes and compete with host protein synthesis. Among the strategies 

viruses use are cleavage of translation initiation factors and associated proteins, manipulating 

translation factors abundance and recruitment into translation initiation complex or expression 

of viral translation factor analogs. Analyzing possible alterations in association of translation 

initiation factors in herpes simplex virus (HSV-1) infected HeLa cells we found that less PABP is 

associated with the cap-binding complex upon infection. Although total PABP amounts were 

unchanged, we observed accumulation of this protein in the nucleus of infected cells in 

contrast with predominantly cytoplasmic localization in mock infected cells. Using GST-PABP 

pull-down and proteomic analyses, we identified several viral proteins interacting with PABP 

including tegument protein UL47 and infected cell protein ICP27. Moreover, we observed 

reduced PABP association with its binding partner PABP interacting protein 2 (Paip2) in HSV-1 

infected cells. Transient expression of ICP27 and UL47 in HeLa cells suggested that UL47 

might play a role in Paip2 displacement from PABP. Co-expression of Renilla luciferase 

reporters with ICP27 and UL47 demonstrated that these viral proteins may affect translation by 

interfering with mRNA nuclear export and/or by activating host cell stress responses. 

112

TARA DOBSON 

113 


Identifying Mechanisms Contributing to (Over)Expression of Aurora A Kinase 

Les Krushel, Tara Dobson 

University of Colorado Denver, School of Medicine, United States of America 

Aurora A kinase activity is essential for mitosis, requiring tight regulation of protein expression 

during the G2/M phase of the cell cycle. Indeed, misregulating Aurora protein levels is 

detrimental to the cell. For example, induced overexpression of Aurora A leads to 

tumorigenesis, while reduced Aurora A kinase expression inhibits cell division and initiates cell 

death. Consequently, agents targeting Aurora A kinase expression could be effective 

chemotherapeutic tools. 

To determine the mechanism by which Aurora A protein levels are enhanced in cancer we 

identified five immortalized cell lines whose protein levels were higher than normal diploid 

fibroblasts yet the mRNA levels were equivalent. This result discounted a transcriptional 

contribution to the overexpression. Moreover the increased Aurora A expression was not due 

to protein stability, indicating that enhanced protein synthesis was responsible. However, the 

proteins that regulate cap-dependent translation eIF-4E and 4E-BP1 were equally expressed 

between the immortalized cells and normal fibroblasts while the amount of 4E-BP1 that was 

hyperphosphorylated was actually higher in the immortalized cells suggesting that there was 

no global increase in cap-dependent translation. Moreover, inhibiting cap-dependent 

translation by knocking down eIF-4E expression or overexpressing a hypophosphorylated 

mutant of 4E-BP1 did not alter Aurora A expression. On the other hand, assays utilizing 

monocistronic and dicistronic RNA constructs revealed that not only does the Aurora A 5’ 

leader contain an IRES but that Aurora A IRES activity is increased in the immortalized cells. 

Taken together, the results suggest that IRES-dependent translation initiation may be a major 

contributor to the normal synthesis of Aurora A and we hypothesize that misregulation of this 

mechanism contributes to the enhanced expression of Aurora A leading to tumorgenesis.



ANKE DOLLER 

PKC delta coordinates RNA-binding and export of nuclear HuR via a dual 

phosphorylation on serine 221 and 318 

Anke Doller 1, Josef Pfeilschifter 2, Wolfgang Eberhardt 2 

1 Hospital of the Johann-Wolfgang-Goethe-University, Germany 

2 Pharmazentrum Frankfurt, Germany 

HuR is a ubiquitous RNA-binding protein that associates with AU-rich element (ARE) bearing 

mRNAs encoding many proinflammatory and proliferative proteins. Although predominantly 

nuclear, HuR translocation to the cytoplasm (HuR-shuttling) is linked to its ability to stabilize 

target mRNAs and modulate their translation. By employing RNA interference technology and 

actinomycin-D experiments, we demonstrate that in human mesangial cells the amplification of 

cytokine-induced COX-2 by Angiotensin II (AngII) occurs via a HuR-mediated increase of 

mRNA stability. Using RNA pulldown assay and EMSA experiments, we furthermore 

demonstrate that the AngII- dependent increase in mRNA stability is preceded by at least two 

well mediated processes including the binding to ARE-bearing target mRNA and the export of 

nuclear HuR-mRNA complex to free and cytoskeletal-bound polysomes indicative for an active 

ribonucleoprotein complex. Mapping of PKC-phosphorylation sites by an in vitro kinase assay 

identified serines 221 and 318 as critical target sites for PKCδ-triggered HuR phosphorylation. 

Mutational analysis revealed that phosphorylation of serine 221 did not affect ARE binding, but 

is indispensable for nuclear- cytoplasmic-shuttling of the HuR-mRNA complex. In contrast, 

mutation of serine 318 although destroying the AngII- induced binding of HuR to the target 

mRNA but did not affect the stimulus-induced nucleo-cytoplasmic HuR-shuttling. Functionally, 

HuR and PKCδ, both are indispensably involved in the AngII-triggered expression of cyclin A, 

D1 and COX-2 and cell migration by AngII. Our data implicate that serine phosphorylation at 

different HuR domains by nuclear PKCδ couples mRNA-binding with nuclear export of HuR. 

Posttranslational modification of HuR by PKCδ represents a novel mode of HuR activation 

implied in renal gene regulation. 

114

VICTORIA DORONINA 

Dissecting ‘stop - carry on translational recoding 

Victoria Doronina, Pamila Sharma, Fu Yan, Anna Tang, Jeremy Brown 

Newcastle University, United Kingdom 

115 


The 19 amino acid “2A” peptide from foot and mouth disease virus has a unique property of 

directing in cis, co-translational separation of nascent polypeptide chains. Previous data 

indicate that the first part of the 2A reaction is an unusual termination catalysed by release 

factors with the final glycine and proline codons of 2A in the ribosomal P and A sites. The 

current model proposes that the N-terminal part of the peptide forms an alpha helix, and that 

interactions of this and the C-terminal conserved motif with the ribosome a) sterically hinder 

formation of the glycine-proline bond encoded by the mRNA and b) direct the ribosome into a 

conformation compatible with RF binding leading to release the peptide from glycyl-tRNA. To 

probe sequence requirements within 2A we have carried out mutagenesis. Consistent with the 

model, mutations that reduce the propensity of the N-terminal portion of the peptide to form an 

alpha helix are generally detrimental. In addition, some mutations that significantly reduce 2A 

activity on their own can be suppressed by further mutations within the peptide, suggesting 

that the conformation/interactions of the peptide as a whole are critical for its function. In 

further mutagenesis experiments codons within the conserved motif were replaced by 

stop-codons. At most positions this led to normal termination events, but translation of mRNAs 

containing stop codons at the final postions of 2A yielded stable peptidyl(2A)-tRNA adducts. 

This has similarity to the action of a number of uORF peptides that stall ribosomes and 

suggests that conformational changes driven by 2A and uORF peptides lead to incompatibility 

between release factors engaging productively with both decoding and peptidyl-transferase 

centres.



JONATHAN DOUGHERTY 

Disruption of processing body (PB) formation by a plus-strand RNA virus 

Jonathan Dougherty, Richard Lloyd 

Baylor College of Medicine, United States of America 

Cytoplasmic mRNA granules such as stress granules (SG) and processing bodies (PB) are 

proposed to be sites of aggregation of translationally silenced mRNA and mRNA degradation 

in eukaryotic cells. We have investigated whether poliovirus (PV), which is a plus strand RNA 

virus containing a genome that is a functional mRNA, may antagonize the processes that lead 

to formation of these structures. Thus, investigation of viral interference in cellular processes 

can provide information about key mechanisms that control SG and PB formation. We have 

previously shown that PV infection inhibits the ability of cells to form stress granules by cleaving 

RasGAP-SH3-binding protein (G3BP) but not Tia-1 or TIAR, implicating G3BP as a key factor 

required for SG formation. Here we show that PB are also disrupted during PV infection in cells 

by 4 hours post infection. The kinetics of PB disruption correlate with production of viral 

proteases. The organizing principle that forms PB foci in cells is unknown, however potential 

scaffolding, aggregating or other types of organizing proteins may be targets of viral proteases. 

We examined the fate of several proteins known to be integral components of PBs and 

determined that two factors involved in 5' end decapping and RNA degradation, Xrn1 and 

Dcp1a, undergo cleavage or accelerated degradation during virus infection. In addition, one 

component of 3' deadenylase complexes, Pan3, was also degraded during infection. Other 

deadenylase components Pan2, Ccr4 and Caf1 were not degraded during infection, as well as 

Rck/p54 helicase. Further investigation revealed that Dcp1 may be a direct substrate of 

poliovirus 3C proteinase. Since it has been shown that deadenylation activity is required for PB 

formation, viral inhibition of deadenylation, through Pan3 degradation, is a potential mechanism 

of P-body disruption. Further experiments are required to determine if loss of scaffolding or 

deadenylation activity are key to disruption of PBs. 

116

JACK DUNKLE 

Structures of the ribosome in an intermediate state of translocation 

Jamie Cate, Jack Dunkle, Wen Zhang 


117 


After each codon of mRNA is translated, the ribosome must move the mRNA and tRNAs, so 

that a new aminoacylated tRNA can enter the ribosome. The process of moving mRNA and 

tRNAs by one codon through the ribosome, termed translocation, is a mechanically 

challenging process for the ribosome. The mRNA and tRNA must be moved by tens of 

Ångstroms in space without the reading frame of the message being disrupted. A necessary 

feature of translocation (Horan, 2007) is rotation of the ribosomal 30S and 50S subunits with 

respect to each other (Frank, 2007). Electron microscopy has revealed some details regarding 

how the ribosome accomplishes this task, but an atomic resolution x-ray crystal structure 

would reveal many more details. We have now solved x-ray crystal structures of the ribosome 

that reveal the structural rearrangements at the ribosomal interface that likely occur during the 

subunit rotation process. Based on the structures, we hypothesize that some intersubunit 

contacts, or bridges, rearrange before others, i.e. the bridges migrate in a sequential, not 

concerted, fashion during mRNA and tRNA translocation. These structures help to explain how 

the ribosome accomplishes translocation in an orderly way during protein synthesis.



OLIVIER DUSS 

NMR Study of the 60kDa Complex between the ncRNA RsmZ and the 

Bacterial Global Regulatory Protein RsmE 

Olivier Duss, Mario Schubert, Frédéric Allain 

ETH Zurich, Switzerland 

In bacteria a class of non-coding RNA (CsrB/RsmZ) regulates translation by sequestering the 

global translational repressor CsrA/RsmE (Babitzke et al., Curr Opin Microbiol., 2007). The 15 

kDa homo-dimeric global bacterial regulatory protein RsmE repress translation in binding to the 

Ribosome Binding Site (RBS) of a subset of mRNAs containing an ANGGA binding consensus 

sequence. The solution structure of the complex between the Shine-Dalgarno sequence (RBS) 

in the 5’UTR of the hcnA mRNA and RsmE has been solved recently in our group and revealed 

the molecular basis of RNA recognition by RsmE (Schubert et al., Nat Struct Mol Biol., 2007). 

However not much is known on how a non-coding RNA containing several ANGGA motifs is 

able to remove the regulatory protein RsmE from the RBS of the mRNA de-repressing 

translation. We therefore started to investigate a complex between the non-coding RNA RsmZ 

(1-89) in complex with one to several RsmE protein dimers by NMR. The non-coding RNA 

RsmZ has four predicted stem-loops (SL1-4) containing an ANGGA motif. 

Based on our current results, we can propose a first model on how RsmE binds the ncRNA 

RsmZ. In a first step, SL2 and SL3 bind RsmE yielding a stable intermediate. In a second step 

SL1 and SL4 bind another RsmE dimer yielding a 60kDa complex. We now aim at determining 

the structure of this complex following a modular approach: first in studying the single 

stem-loops of this RNA in complex with the protein dimer and second in constructing a model 

of the whole complex using RDCs, PRE and segmental isotope labeling of the RNA (Lukavsky 

et al., Methods Enzymol., 2005). We will report here on the solved NMR structures of SL1, SL2 

and SL3 of RsmZ in complex with the RsmE dimer. The structures explain the fine-tuning of 

the binding affinity to RsmE depending on the specific RNA sequence of the stem-loop. 

118

NAAMA ELDAD 

119 


RNA Polymerase II subunits link transcription and mRNA decay to translation 

Technion, Israel 

Little is known about cross talk between transcription and translation machineries that operate 

in the different eukaryotic compartments. The yeast Rpb4/7 heterodimer shuttles between the 

nucleus, where it functions in transcription, as a part of RNA Polymerase II (Pol II), and the 

cytoplasm, where it functions in the major mRNA decay pathways. Here we show that Rpb4/7 

interacts physically and functionally with components of the translation initiation factor 3 (eIF3) 

in RNA independent manner. Using various approaches (drug sensitivity test, methionine 

incorporation rate and polysomal separation through sucrose gradient), we show that Rpb4/7 

is required for efficient translation initiation and for transport of translationally repressed mRNPs 

from processing bodies (P-bodies) to polysomes. Remarkably, efficient translation depends on 

association of Rpb4/7 with Pol II in the nucleus. Thus, Pol II controls translation in the 

cytoplasm, suggesting the first time in eukaryotes, a coupling between transcription and 

translation events.



IRINA ELISEEVA 

On the mechanism of YB-1 mRNA translation inhibition by polyadenylation 

Irina Eliseeva, Dmitry Lyabin, Lev Ovchinnikov 

Institute of Protein Research, Russian Federation 

The eukaryotic multifunctional protein YB-1 is involved in almost all DNA- and 

mRNA-dependent events in the cell. As we showed previously, YB-1 synthesis in vitro is under 

negative control of YB-1 and positive control of PABP that compete with each other for the 

overlapping specific binding sites in a regulatory sequence of the 3′ UTR of YB-1 mRNA [1,2]. 

Also, it was found that polyadenylation of YB-1 mRNA decreases its translational activity 

presumably due to formation of a cyclic structure at its 3′ terminus that comprises the poly(A) 

tail, some proteins of reticulocyte lysate, and the regulatory sequence. 

Here, we report that the inhibitory effect of the poly(A) tail on YB-1 mRNA translation is 

dependent on the length of the spacer between the regulatory sequence and the poly(A) tail, 

and independent of its nucleotide sequence, and that this effect is observed for capped mRNA 

only. Unlike in controls where luciferase poly(А)+ mRNA binds much more eIF4G than its 

poly(А)- form, the both forms of YB-1 mRNA bind the same amount of eIF4G. Additionally, we 

showed that the 3′ UTR fragment of YB-1 mRNA specifically binds eIF4G from reticulocyte 

lysate in a PABP-independent manner. 

These experimental results will be helpful in describing the mechanism of regulation of YB-1 

mRNA translation. 

This study was supported by RFBR (#07-04-00403-а) and by the RAS Programs on 

"Molecular and Cellular Biology" and "Basic Science - to Medicine". 

Skabkina, OV et al. (2003) J Biol Chem 278:18191-18198. 

Skabkina, OV et al. (2005) Mol Cell Biol 25:3317-3323. 

120

KEI ENDO 

121 


Post-transcriptional gene repression induced by an artificial cis element of RNA 

aptamer to mammalian initiation factor eIF4AIII 

Kei Endo, Yoshikazu Nakamura 

Institute of Medical Science, University of Tokyo, Japan 

To control transgene expression at will is one of the great challenges in biotechnology and 

biomedical engineering, and has become increasingly important. Transcription has 

fundamental effects on gene expression, but recent studies revealed the importance of 

post-transcriptional regulation. In contrast to transcriptional control, however, tools for 

post-transcriptional control remain largely undeveloped. From this viewpoint, we expect RNA 

aptamers, which are capable of capturing their targets, can become powerful tools as cis 

regulatory elements on mRNA. In this study we developed RNA aptamer to mammalian 

initiation factor eIF4AIII, and examined for its capacity to control transgene expression as a 

regulatory cis-element. eIF4AIII is known as a core factor of the Exon Junction Complex (EJC), 

which is recruited to mRNA in a splicing dependent manner and acts as an intermediary 

between post-transcriptional processes. The EJC in the 3' UTR down-regulates the gene 

expression by a process called nonsense mediated mRNA decay (NMD). RNA aptamer to 

eIF4AIII was selected by SELEX procedure. It binds specifically to eIF4AIII, but not to eIF4AI, 

and does not interfere with the known interaction of eIF4AIII with other cellular components 

such as eIF4G, Y14, Magoh, and MLN51, nor the ATP hydrolysis activity of eIF4AIII. 

Nevertheless, when the aptamer sequence was inserted in the 3' UTR of a reporter gene, it 

greatly reduced the reporter expression in cultured mammalian cells. These findings suggest a 

splicing-independent reconstitution of the EJC leading to NMD, or a novel translational 

repression independent of the EJC. These and other attempts of constructing cis-acting 

regulatory elements with RNA aptamers to translation initiation factors will be presented.



IVAN FEDYUNIN 

Impact of altered translation by modified tRNA-profile on the co-translational 

folding 

Ivan Fedyunin, Gong Zhang, Zoya Ignatova 

Potsdam University, Germany 

The nucleotide sequence (mRNA) is not an inert structure and can also provide an additional 

structural information to kinetically fine-tune protein biogenesis (i.e., protein abundance, folding 

fidelity). The speed of translation along mRNA is non-uniform and is determined by the 

concentration of tRNAs that pair to the corresponding codons, determining thus the speed 

each codon is read. Analysis of proteome-wide scale in E. coli revealed that local acceleration 

of the protein elongation by up-regulation of some low-abundant tRNAs increased the 

aggregation propensity of proteins with a rough translation profile undergoing through several 

local slow-translating minima. The level of up-regulated tRNAs was quantified with both, 2D-gel 

electrophoresis and RT-PCR. In addition, the cell growth is significantly slowed down when 

low-abundant tRNAs are up-regulated, whereas there is no difference in cell growth at 240C 

when global rate of translation is slowed down. These results indicate the significance of the 

non-uniform translational kinetics for co-translational protein folding on a proteome-wide scale 

and the importance of fine-tuning tRNA-profile to maintain cell viability. 

122

MEGAN FILBIN 

123 


Importance of Long-Range Communication Between Two Domains in the HCV 

IRES for Formation and Fidelity of 80S Ribosomes 

Megan Filbin, Bradley Nelson, Jeffrey Kieft 

University of Colorado Denver, Anschutz Medical Campus, United States of America 

During the first major step in infection, Hepatitis C virus (HCV) uses a highly structured internal 

ribosome entry site (IRES) to translate its genome. The IRES RNA consists of three major 

domains (II-IV) spanning approximately 340 nucleotides that specifically bind eukaryotic 

initiation factor (eIF) 3 and the 40S ribosomal subunit. Domain II of the IRES RNA lies near the 

E-site of the 40S ribosomal subunit and is important for the release of eIF2 and 80S formation, 

however the mechanism by which it does this is unknown. We hypothesize domain II plays a 

specific role via intramolecular communication with domain IV in orienting the AUG start codon 

in the P-site of the 40S ribosomal subunit, a necessary step for 80S formation that normally 

requires scanning and the presence of the Kozak sequence. To test this idea, we have used 

selective 2’ hydroxyl acylation analyzed by primer extension (SHAPE) in a novel way to probe 

the structure of the IRES within preinitiation complexes. SHAPE reveals that domain II might 

have a role in the unwinding of the AUG start codon-containing domain IV stem-loop and 

maintaining the AUG in the decoding groove of the 40S subunit. Mutations made in the apex 

of domain II can form 80S complexes initially, however these mutants are defective in their 

ability to initiate translation. Hence, 80S formation by the HCV IRES is decoupled from 

successful progression to elongation. Toeprinting analysis reveals that the inability to translate 

a downstream reporter may be because the AUG is not properly docked into the 40S 

decoding groove, even though it can form 80S ribosomes. Therefore, domain II appears to be 

specifically important for not only proper 40S-IRES interaction resulting in eIF2 release and 80S 

ribosome formation, but also for events that occur between 80S formation and the start of 

elongation.



JEFFREY FISCHER 

Mechanistic insights into the function of the universally conserved GTPASE 

HFLX from escherichia coli 

Jeffrey Fischer, Michael Shields, Hans-Joachim Wieden 

University of Lethbridge, Canada 

Protein synthesis is a highly conserved process in all living cells involving several members of 

the TRAFAC class of P-loop GTPases, which play essential roles during translation. The 

universally conserved GTPase HflX has previously been shown to associate with the 50S 

ribosomal subunit, as well as to bind and hydrolyze both GTP and ATP. In an effort to elucidate 

the cellular function of HflX, we have determined the kinetic parameters governing the 

interaction between HflX and these two purine nucleotides using fluorescence based 

steady-state and pre-steady state techniques. Based on the obtained kinetic parameters, we 

demonstrate that the GTPase activity of HflX is stimulated by 50S ribosomal subunits, as well 

as by empty and poly(U) programmed 70S ribosomes. Interestingly, the 70S stimulated 

GTPase activity is specifically inhibited by the antibiotic chloramphenicol, which binds to the 

large ribosomal subunit, but not by kanamycin, an aminoglycoside targeting the small 

ribosomal subunit. 

124

JOHN FLANAGAN 

125 


Observation of distinct A/P hybrid-state tRNAs in translocating ribosomes 

Olivier Namy 1, John Flanagan 2 

1 Institut de Genetique et Microbiologie, France 

2 University of Oxford, United Kingdom 

During translocation, tRNAs move between ribosomal subunits from aminoacyl (A) site to 

peptidyl (P) site, and from P-site to exit (E) site, a series of ‘hybrid’ intermediate states. Some 

intermediate states occur spontaneously and some appear to require presence of the 

translocase enzyme EF2 (in eukaryotes, EF-G in prokaryotes). The translocase is essential for 

productive protein synthesis, but some tRNA movement can be fostered by the ribosome 

itself, or occurs spontaneously. We are studying ribosome translocation by determining 

structures of intermediate states purified from translation reactions and stalled on mRNA 

secondary structures. We have shown that a population of mammalian ribosomes stalled at an 

mRNA pseudoknot structure contains structurally-distorted tRNAs in two different A/P hybrid 

states. In one, which we denote A/P’, the tRNA is in contact with the translocase EF-2 which 

induces it. In the other, called A/P’’, the translocase is absent. The existence of these 

alternative A/P intermediate states has relevance to our understanding of the mechanics and 

kinetics of translocation, and provides for a structural understanding of single molecule kinetic 

studies of ribosome movement.



PAUL FOX 

Cdk5- and p70 S6K-mediated, 2-site phosphorylation of a tRNA synthetase 

induces transcript-selective inhibition of translation 

Paul Fox, Abul Arif 

Cleveland Clinic, United States of America 

Interferon (IFN)-gamma induces assembly of the heterotetrameric GAIT (Gamma-interferon 

Activated Inhibitor of Translation) complex consisting of the bifunctional glutamyl-prolyl tRNA 

synthetase (EPRS), ribosomal protein L13a, NS1-associated protein-1 (NSAP1), and GAPDH. 

The complex binds a structural element in the 3’UTR of multiple inflammatory transcripts and 

silences their translation in myeloid cells. Mass spectrometric analysis revealed IFN-gamma 

induces phosphorylation at Ser886 and Ser999 in the non-catalytic linker joining the synthetase 

domains of human EPRS. These phosphorylation events orchestrate the stepwise release of 

EPRS from the parental multi-aminoacyl tRNA synthetase complex, assembly of the GAIT 

complex, mRNA binding, and translational silencing activity. Taking advantage of bioinformatic, 

biochemical, and genetic approaches, we have identified a network of IFN-gamma-activated 

kinases that coordinates the two-step phosphorylation and consequent activation of EPRS. 

Cyclin-dependent kinase 5 (Cdk5), in conjunction with its regulatory protein Cdk5R1 (p35), 

induces the initial phosphorylation of EPRS at Ser886. Subsequently Cdk5/p35, in coordination 

with mTORC1, phosphorylates and activates 70 kDa ribosomal protein S6 kinase (S6K), which 

in turn phosphorylates EPRS at Ser999 to generate functional, translation silencing-competent 

EPRS. In previous reports, anabolic signals caused S6K-mediated activation of the protein 

synthetic apparatus to stimulate translation globally. In contrast, we find that an inflammatory 

stimulus induces S6K-mediated phosphorylation of an mRNA-binding protein for 

transcript-selective inhibition of translation. 

126

MAGALI FRUGIER 

127 


Particularities of Plasmodial translational machinery: Structural and functional 

analysis of specific insertions in Plasmodium falciparum Aspartyl-tRNA 

synthetase 

Magali Frugier, Tania Bour, Bernard Lorber, Richard Giegé 

UPR ARN du CNRS, France 

Distinctive features of aminoacyl-transfer RNA (tRNA) synthetases (aaRS) from the protozoan 

Plasmodium genus are described. We have identified 36 aaRS genes in the P. falciparum 

genome. Encoded proteins are dedicated to both cytosolic and apicoplastic translations. 

Sequence alignments of Plasmodium aaRSs with their analogues (Homo sapiens and 

Saccharomyces cerevisiae) reveal strong differences. Indeed, the presence of many extensions 

and insertions characterizes the parasite enzymes. In general these additional domains are 

conserved in all Plasmodia, their presence does not correlate with aaRS classification, 

oligomerization or with their putative cellular localizations. 

The plasmodial AspRSs display an insertion in their anticodon-binding domains and a 

remarkably long N-terminal appendix that varies in size from 110 to 165 aa, which contains 

two conserved potential initiation codons. We focused on the protein from Plasmodium 

falciparum, the causative parasite of human malaria. In this specie, AspRS is 626 or 577 amino 

acid-long, depending on whether initiation starts on the first or second in-frame initiation 

codon. The longer protein has poor solubility and propensity to aggregate. The recombinant 

short version migrates with endogenous AspRS and thus was priviledged in this study. 

Comparison of the tRNA aminoacylation activity of wild-type and mutant P. falciparum 

AspRSs with functionality of the yeast and human AspRSs revealed unique properties. The 

N-terminal extension contains a motif that provides unexpectedly strong RNA binding capacity 

to the plasmodial AspRS. Further, experiments demonstrate the requirement of the plasmodial 

insertion for AspRS dimerization, hence for tRNA aminoacylation and putative other functions. 

These data provide a robust background for unraveling the precise functioning of the parasite 

translation and for developing novel lead compounds against malaria targeting its idiosyncratic 

domains.



CHIARA GAMBERI 

Bicaudal-C and Ccr4 repress nanos expression during oogenesis 

Chiara Gamberi, Paul Lasko 


Bicaudal-C (Bic-C) functions to establish anterior-posterior polarity in the oocyte via repression 

of mRNA translation. In wild-type oocytes, Nanos (Nos) protein is restricted to the pole plasm 

through multiple levels of translational repression. We found that Bic-C binds to nos mRNA 

both in vitro and in vivo and participates in regulating nos translation. Unlike wild-type ovaries, 

where Nos is only detectable in the germarium and at the end of oogenesis, in Bic-C 

homozygous mutants Nos protein accumulates in the oocyte, starting as early as stage 4, and 

is also detectable in the nurse cells. In mid-oogenesis, the average polyA tail length of nos 

mRNA in Bic-C ovaries is greater than in wild-type controls. We also observe this in twin (ccr4) 

mutant ovaries, suggesting that Bic-C and CCR4 may regulate nos expression via 

deadenylation, as has been previously established for Bic-C mRNA. Bic-C 

co-immunoprecipitates with Glorund (Glo), another nos repressor, and this interaction is almost 

completely dependent on the presence of RNA, suggesting that they co-exist in the same nos 

ribonucleoprotein complexes. Embryos produced by Bic-C/+ heterozygous mothers exhibit 

ectopic Nos, which may interfere with anterior morphogenesis. Therefore, Bic-C dependent 

Nos derepression may underlie the observed bicaudal phenotype. 

128

NIELS GEHRING 

129 


Disassembly of exon junction complexes by the ribosome-associated protein 

PYM 

Niels Gehring 1, Styliani Lamprinaki 1, Matthias Hentze 1, Andreas E. Kulozik 2 

1 EMBL, Germany 

2 MMPU EMBL/University of Heidelberg, Germany 

During processing of newly transcribed RNAs in the nucleus, the spliceosome loads 

exon-junction complexes (EJC) onto spliced mRNAs. These EJCs play a decisive role in 

nonsense-mediated mRNA decay (NMD) and enhance translation and polysome association in 

mammalian cells. 

The removal and recycling of EJCs from mRNAs have been attributed to ribosomal passage 

during translation. We have identified the protein PYM as an EJC disassembly factor. EJCs fully 

assembled by in vitro splicing are efficiently disassembled by PYM, a function that is abolished 

when the direct interaction between PYM and the MAGOH-Y14 heterodimer, a structural 

component of EJCs, is impaired. 

PYM overexpression in cells decreases the association of EJCs with spliced mRNA and 

inhibits nonsense-mediated mRNA decay. Conversely, EJCs accumulate on spliced mRNAs in 

cells depleted of PYM. Hence, PYM acts as an EJC disassembly factor both in vitro and in 

living cells and can antagonize important EJC-dependent functions such as NMD (Gehring et 

al., 2009). 

Within cells, translation-independent disassembly of EJCs by PYM appears to be prevented by 

its stable association with ribosomes, which restricts the activity of PYM to mRNAs that are 

translated. Notably, HeLa cells express far fewer molecules of PYM than the number of 

ribosomes per cell. This raises the interesting question of whether and how PYM associates 

preferentially with the ribosomes that execute translation, or whether ribosomes bound by PYM 

are preferentially selected for translation. Hence, PYM may functionally link stimulation of 

translation and the EJC. These aspects of PYM regulation and its potential function in 

translation stimulation are currently under experimental investigation. 

Gehring, N.H., Lamprinaki, S., Kulozik, A.E:, Hentze, M.W. (2009) Disassembly of exon junction 

complexes by PYM. Cell,137, 536-48.



ROBERT GILBERT 

Eukaryotic ribosome structure in the light of cellular differentiation 

Robert Gilbert, Luke Yates 

University of Oxford, United Kingdom 

We are using cryo-electron microscopy to study the structures of ribosomes while actively 

engaged in translation purified from in vitro translation reactions and growing cells, in order to 

study the developmental regulation of ribosome composition and structure. As part of these 

projects, we have purified translating complexes from the human cell lines 293T and HeLa and 

from the slime mould Dictyostelium discoideum in mid-log growth phase. Reconstruction of the 

human ribosomes provides a basis for describing differences between their structures in 

different states and compared to previously-obtained data. Reconstruction of the Dictyostelium 

ribosomes pinpoints the physical location of expansion segments in the rRNA of this primitive 

eukaryotic model organism. Altogether these studies show how cell biology and ribosomal 

activity can be correlated to one another, and are allowing us to investigate the physical 

correlates of reports in the literature that ribosomal composition varies markedly with cell type. 

130

CHRISTIAN GOETZ 

eIF4E Status Controls cap-Independent Translation and Oncolysis of a 

Poliovirus Recombinant 

Christian Goetz, Richard Everson, Linda Zhang, Matthias Gromeier 


131 


Translation initiation is tightly regulated by a variety of stimuli originating from the intracellular or 

extracellular environment. One of these controls occurs in the form of growth signaling 

pathways that phosphorylate key molecules in the initiation machinery. The Pi3k/mTor pathway 

phosphorylates 4E-BPs leading to disengagement of 4E-BPs from eIF4E and subsequent 

binding of eIF4E to eIF4G. The Ras pathway and stress response pathways, mediated by p38, 

cause the activation of the eIF4E kinase MNK resulting in phopshorylation of eIF4E . The 

effects of this eIF4E modifictaion on translation are poorly understood. 

By taking advantage of a cell type specific poliovirus chimera, relying purely on 

cap-independent mechanisms for translation initiation, we show that both signaling pathways 

influence the balance between cap-dependent and cap-independent translation initiation. 

Inhibition of the MNK kinase decreases viral proliferation, translation and oncolysis in a naturally 

permissive glioma cell line while Pi3k inhibition stimulates viral growth and translation. Further, 

we show that this control is exerted at the level of eIF4E. Thus eIF4E integrates the signals 

from two major growth signaling pathways by regulating translation initiation of specific 

mRNAs.



ANNA GOLOVINA 

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that 

specifically modifies A37 of tRNA1Val (cmo5UAC) 

Anna Golovina 1, Andrey Golovin 1, Alexey Bogdanov 1, Olga Dontsova 1, Petr Sergiev 1, 

Marina Serebryakova 2, Irina Demina 2, Vadim Govorun 2 

1 Moscow State University, Russian Federation 

2 Proteome Center of the Russian Academy of Medical Sciences, Russian Federation 

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various 

modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo5UAC) contains a 

unique modification, N6-methyladenosine, at position 37, however the enzyme responsible for 

this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an 

enzyme responsible for the methylation of A37 in tRNA1Val. Inactivation of yfiC gene abolishes 

m6A formation in tRNA1Val, while expression of the yfiC gene from a plasmid restores the 

modification. Additionally, unmodified tRNA1Val can be methylated by recombinant YfiC 

protein in vitro. Although the methylation of m6A in tRNA1Val by YfiC has little influence on the 

cell growth under standard conditions, the yfiC gene confers a growth advantage under 

conditions of osmotic and oxidative stress. 

(The paper is currently in press in RNA journal) 

132

BARBARA GORGONI 

133 


Multiple Poly(A)-Binding Proteins are essential for Xenopus laevis development 

Nicola Gray 1, Barbara Gorgoni 2, William Richardson 2, Gavin Wilkie 2, Hannah Burgess 2, 

Matthew Brook 2, Michael Sheets 3 

1 Medical Research Council, United Kingdom 

2 MRC-HRSU and University of Edinburgh, Germany 

3 University of Wisconsin School of Medicine and Public Health, United States of America 

Translation of specific mRNAs during gametogenesis and early embryogenesis is regulated by 

alterations in poly(A) tail length. The function of poly(A) tails is mediated by a family of 

poly(A)-binding proteins (PABPs). In Xenopus laevis, three cytoplasmic PABPs have been 

described: PABP1, ePABP and ePABP2. Both PABP1 and ePABP stimulate translation while 

ePABP2 is reminiscent of nuclear PABP. More recently our group identified a PABP that 

segregates closely with mammalian PABP4 and that appears to be structurally and functionally 

similar to PABP1 and ePABP. Interestingly, the different PABPs are expressed simultaneously 

in several adult and embryonic tissues. This raises the fundamental question as to whether 

they are functionally redundant or have individual roles. 

To address this issue we have selectively knocked-down PABP1, ePABP and PABP4 in 

Xenopus embryos, providing the first vertebrate PABP phenotypes. PABP1 and ePABP 

morpholino-mediated knock-downs show severe early phenotypes, with defects in both 

anterior and posterior structures, and are embryonic lethal. PABP4 morphants show a very 

different phenotype that arises later during development and appears to affect only anterior 

structures. These results reveal essential roles in development for each of the PABP proteins. 

To further investigate the molecular basis of this specificity, we have compared the ability of 

different PABPs to rescue the PABP1 phenotype. Our results support the hypothesis that 

structurally similar PABP proteins have both overlapping and distinct roles in development.



STEFANO GROSSO 

mTORc1 inhibition does not repress initiation of translation in cancer cell 

models 

Stefano Grosso 1, Daniela Brina 1, Claudio Procaccianti 1, Elisa Pesce 1, Odette Baldi 1, 

Stefano Biffo 1, Elia Ranzato 2, Pier Carlo Marchisio 3 

1 DIBIT, San Raffaele Scientific Institute, Italy 

2 DISAV, University of Piemonte Orientale, Italy 

3 Università Vita- Salute San Raffaele, Italy 

Eukaryotic Initiation Factor 4F (eIF4F) is composed of the cap-binding protein eIF4E, the 

scaffold protein eIF4G and the ATP-dependent helicase eIF4A, which, assisted by eIF4B/H, 

unwinds secondary structures in the 5’ UTR of the mRNA. The activity of eIF4F complex 

increases translation of most capped, polyadenylated mRNAs and translation of 5'UTR 

structured mRNAs. Many mRNAs involved in cell cycle progression and tumorigenesis have a 

structured 5'UTR and consequently, indirect inhibition of eIF4F is crucial in cancer therapy. 

eIF4F is controlled by the mTORc1 pathway that is activated by growth factors and optimal 

nutrient conditions. Rapamycin blocks the formation of the eIF4F complex by inhibiting mTOR. 

However, in several cancer cells, mTOR inhibition only marginally blocks the translation rate, 

suggesting that other pathways are important. For example, we recently described that 

RACK1/PKCβII complex can affect translation directly, in an mTOR independent fashion, 

acting close to the ribosomal machinery through the scaffolding activity of RACK1 on the 

ribosome. We further investigated mTOR independent translation in cancer cells. Malignant 

mesothelioma (MM), a fatal type of tumor arising from pleura and peritoneum, is associated 

with exposure to asbestos. MM cell line (REN) was grown in presence or absence of growth 

factors, in order to study regulation of translation. We found growth factor independent 

translation in REN cells. Next we dissected the pathway regulating protein synthesis. 

Pharmacological inhibition of mTOR and MAP kinase pathways, resulted in dephosphorylation 

of molecular targets, but protein synthesis was not affected, suggesting that deregulation of 

protein synthesis may occur at the level of cap complex formation. We will further address 

whether different classes of mRNAs are translated in REN cells and in other models of mTOR 

independent translation. 

134

GABRIEL GUARNEROS 

135 


Translation termination factors (RF1, RF2, and RF3), ribosome recycling factor 

(RRF) and elongation factor (EF-G) rescue ribosomes stalled at sense codons 

Gabriel Guarneros 1, Serafín Vivanco-Domínguez 1, José Bueno-Martínez 1, Marco Antonio 

Magos-Castro 1, Gloria León-Avila 2, Nobuhiro Iwakura 3, Hideko Kaji 3, Akira Kaji 4 

1 CINVESTAV, United States of America 

2 ENCB, Mexico 

3 Thomas Jefferson University, United States of America 

4 University of Pennsylvania, United States of America 

In bacteria, translation of mRNA ends when the ribosome reaches a stop codon, the mature 

protein is released and the translational machinery dissociated into free components: 

ribosomal subunits, mRNA and the last used tRNA. This process, which involves factors RF1, 

RF2, RF3, RRF and EF-G, allows the ribosome components to be assembled again and start a 

new cycle of protein synthesis. It is known that ribosome often stalls on mRNA for various 

reasons. To study whether termination factors help cells rescue the stalled ribosomes we 

induced ribosomal stalling at particular sense codons located early or late in mRNA by 

starvation for specific aminoacyl tRNAs. Then, the contribution of RF1, RF2, RF3 and RRF 

proteins to release the stalled ribosomes was analyzed by using mutants defective in each 

factor. The results showed that, under limitation of each factor, the ribosome stalled in 

complexes containing mRNA and peptidyl-tRNAs of the appropriate size and specificity. In 

vitro experiments using native ribosome with nascent peptidyl tRNA as substrates mimicking 

stalled ribosomes, showed that RF1 or RF2 in combination with RF3, RRF and EF-G 

disassembled the complex. RF1 or RF2 with RF3 released peptide from the native ribosomal 

complex in the presence of EF-G and GTP. These results show that the termination factors 

and RRF/EF-G participate in a novel mechanism of release of ribosomes stalled at sense 

codons in addition to their classical role in translation termination. This occurs with ribosomes 

stalled at different positions in the mRNA. We believe that this mechanism of ribosome rescue 

helps to maintain protein synthesis and cell viability.



HUILI GUO 

Investigating the effect of microRNAs on ribosome occupancy 

Nicholas Ingolia 1, Jonathan Weissman 1, Huili Guo 2, David Bartel 2 

1 Department of Cellular and Molecular Pharmacology, University of California, San 

Francisco, United States of America 

2 Whitehead Institute for Biomedical Research; Department of Biology, Massachusetts 

Institute of Technology, United States of America 

In animals, microRNAs (miRNAs) are known to regulate gene expression via translational 

repression and/or mRNA destabilization. While the latter has been studied extensively at a 

genome-wide level through the use of microarrays, much less has been done to probe 

translational repression on a similar scale even though proteins are the most relevant readout 

of regulatory function. Recent use of quantitative mass spectrometry-based techniques has 

contributed to our understanding of repression at the protein level, although such techniques 

can be biased towards detecting highly abundant proteins and yield smaller datasets 

compared to genomics approaches. Here, we use ribosome footprinting as a means of 

detecting miRNA-mediated translation repression on a genomic-wide scale. This technique 

combines the scale of genomics with the physiological relevance of proteomics. 

We have performed ribosome footprinting and RNA-seq experiments in parallel in two 

systems: siRNA-transfected HeLa cells, and in vitro differentiated neutrophils derived from 

miR-223 knock-out mice. Ribosome footprinting is able to detect ribosome occupancy 

changes caused by miRNA targeting, thus comparison of the readouts from footprinting and 

RNA-seq should enable us to detect additional impact exerted by miRNAs at the level of 

translation. In addition, this technique captures characteristics of translation at nucleotide 

resolution and should help elucidate mechanistic aspects of miRNA-mediated translation 

repression. 

136

GABRIELLE HAAS 

137 


Analysis of the interactions and P-body localization of decapping activators in 

metazoa 

Gabrielle Haas, Jörg Braun, Sigrun Helms, Izaurralde Elisa, Cátia Igreja 


In eukaryotes, removal of the mRNA 5′-cap structure is catalyzed by the decapping enzyme 

DCP2; but to be fully active and/or stable, DCP2 requires additional proteins including DCP1, 

EDC3, EDC4, HPat and RCK/Me31B. Apart from EDC4, these proteins are conserved, 

nevertheless, information regarding their interactions stems mainly from studies in S. 

cerevisiae. Here, we have characterized the decapping interaction network in D. melanogaster 

and uncovered interactions that have not been reported in yeast. First, DCP1 interacts with 

XRN1; probably ensuring that decapped mRNAs are immediately degraded. Second, HPat 

and DCP1 self-interact in an RNA-independent manner. Third, HPat competes with EDC3 and 

Tral for binding to Me31B, indicating that Me31B assembles into at least three distinct 

complexes. We have mapped the protein domains required for these interactions as well as 

those required for P-body localization. Progress in these experiments will be reported, as will 

efforts to generate protein mutants that no longer interact with each other.



VASILI HAURYLIUK 

Interaction framework among eRF1 / eRF3 / PABP and G nucleotides: 

complete thermodynamical analysis 

Gemma C. Atkinson 1, Ludmila Frolova 2, Vladimir A. Mitkevich 2, Artem V. Kononenko 2, 

Alexander A. Makarov 2, Vasili Hauryliuk 3, Tanel Tenson 3 

1 Department of Evolution, Genomics and Systematics, Uppsala University, Sweden 

2 Engelhardt Institute of Molecular Biology, Russian Academy of Science, Russian 

Federation 

3 University of Tartu, Institute of Technology, Estonia 

Translation termination in eukaryotes is facilitated by concerted action of eRF1 and eRF3 

factors. eRF1 recognizes the stop codon in the A site and promotes nascent peptide chain 

release, and GTPase eRF3 facilitates peptide release via its interaction with eRF1. In addition to 

its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay 

through its association with cytoplasmic poly(A) binding protein (PABP) through PAM2-1 and 

PAM2-2 motifs [1]. This interaction is considered as a regulator of the PABP interaction with 

the 3’-poly(A) tail of mRNAs, suggesting that eRF3 has an important role in the mRNA 

degradation and/or the regulation of translation efficiency [2]. All available biochemical data on 

the eRF3 GTPase cycle were obtained using an eRF3 variant lacking the N domain. 

Preparation of full-length eRF3 was never achieved, although the truncated version of the 

factor was first purified almost 15 years ago [3]. We have for the first time studied complex 

formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using 

isothermal titration calorimetry, and demonstrated formation of eRF1:eRF3:PABP:GTP 

complex. Analysis the temperature dependence of eRF3 interactions revealed major structural 

rearrangements accompanying formation of eRF1:eRF3:GTP, as opposed to eRF1:eRF3:GDP, 

in agreement with the established active role of GTP in promoting translation termination. Using 

point mutagenesis we have shown that PAM2-2, but not PAM2-1 as was suggested earlier [5] 

is indispensible for eRF3:PABP complex formation. This provides a tool for dissecting the roles 

of eRF3 in translation termination and NMD. [1] Hosoda, N. et al. (2003) J Biol Chem 278, 

38287-91. [2] Amrani, N. et al. (2008) Nature. [3] Zhouravleva, G. et al. (1995) <strong>EMBO</strong> J 14, 

4065-4072. [4] Atkinson, G.C. et al. (2008) BMC Evol Biol 8, 290. [5] Singh, G. et al. (2008) 

Plos Biology 6, 860-871. 

138

VALÉRIE HEURGUÉ-HAMARD 

139 


Function of eRF1 methyltransferase subunits Mtq2p and Trm112p in ribosome 

biogenesis in S. cerevisiae 

Valérie Heurgué-Hamard 1, Sabine Figaro 2, Rémi Mongeard 2, Richard Buckingham 2, 

Georges Lutfalla 3 

1 CNRS, IBPC, France 

2 CNRS, UPR 9073, France 

3 UMR 5235, Université Montpellier II, France 

Stop codons in mRNA are recognised on the ribosome in yeast and mammalian cells by the 

heterodimer eRF1-eRF3, and in eubacteria by RF1 and RF2. Bacterial release factors (RFs) 

evolved independently from yeast and mammalian RFs, and eRF1 possesses only one motif in 

common with them, a GGQ tripeptide that interacts with the peptidyl transferase center of the 

large ribosomal subunit. Strikingly, a post-translational N5-methylation on the Gln residue is 

also conserved. 

S. cerevisiae eRF1, in complex with eRF3, is methylated by the heterodimer Mtq2p-Trm112p. 

Mtq2p carries the catalytic site involved in SAM fixation and methyl transfer to Gln of the GGQ 

motif. Trm112p is a small zinc finger protein, which besides its function in eRF1 methylation, is 

essential for tRNA methylation by Trm9p and Trm11p. Deletion of MTQ2 or TRM112 strongly 

inhibits growth. 

We show here that both components of the eRF1 methyltransferase are involved in ribosome 

biogenesis, probably by two independant and distinct mechanisms. Deletion of MTQ2 or 

inactivation of the catalytic site leads to a defect in 60S ribosomal subunit biogenesis. 

Experiments showed that Mtq2p is not associated with ribosomes, and that inactivation leads 

neither to early defects in rRNA maturation nor to defects in ribosomal export. The role of 

Mtq2p in large ribosomal subunit synthesis depends on its catalytic activity although no new 

substrate has so far been identified. 

TRM112 null cells have reduced levels of small ribosomal subunits that leads to the 

accumulation of free large ribosomal subunits. The same phenomenon is observed on 

overproduction of a partner of Trm112p, such as Mtq2p in its active or inactive state, Trm9p or 

Trm11p. Co-expression of Trm112p restores at least partially 40S biogenesis. Experiments are 

ongoing to test the hypothesis that overproduction of these partners sequester Trm112p, 

preventing its interaction with components involved in 40S biogenesis.



JULIANE HIRNET 

Cap-independent translation of poliovirus is affected by a neuron-specific 

microRNA 

Dagmar Goergen 1, Michael Niepmann 1, Juliane Hirnet 2 

1 Institute of Biochemistry, Germany 

2 Justus Liebig University, Germany 

Poliovirus (PV) infects preferentially neuronal cells and causes poliomyelitis, a severe 

motor-neuronal disease. The preference for neuronal cells is on the one hand due to the PV 

receptor CD155, on the other hand there may be tissue specific factors involved in the tropism 

of PV that act after infection. 

PV belongs to the family of picornaviridae and has a single stranded RNA genome of positive 

polarity. PV translation is initiated cap independently via an internal ribosome entry site (IRES). 

PV IRES dependent translation depends on several cellular factors, the eukaryotic initiation 

factors (eIFs) and IRES trans-acting factors (ITAFs) like the polypyrimidine tract binding protein. 

All these factors are present in most tissues and are therefore unlikely to cause a preference of 

PV to neuronal cells. Consequently, we tried to identify other intracellular factors possibly 

involved in the tissue specificity of PV. 

miRNAs are small non-coding RNAs usually involved in translation repression of their target 

mRNAs. Here we investigated the influence of miRNAs specific for neuronal cells on translation 

initiation via the PV IRES. miRNA 326 and 127 were identified to bind the PV IRES. Their 

influence on translation efficiency of PV was investigated using luciferase reporters in the cell 

free rabbit reticulocyte lysate and via RNA transfection into HeLa and SH-SY5Y neuronal cells. 

No effect could be seen using miRNA127. However, addition of miRNA 326 duplex lead to a 

significant dose dependent increase in translation efficiency when transfected into cells. The 

effect was less pronounced In SH-SY5Y neuroblastoma cells, perhaps due to endogenous 

miRNA326. The stimulatory effect of miRNA326 on PV translation contrasts the well-known 

effects of miRNAs which usually inhibit cellular translation or even target RNAs for degradation. 

140

PHILIP HOWE 

Transforming Growth Factor-ß (TGFß)-mediated Transcript Selective 

Translational Activation of EMT Inducer mRNAs 

Philip Howe, Arindam Chaudhury, George Hussey, Paul Fox 

Cleveland Clinic, United States of America 

141 


Transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transdifferentiation (EMT) 

is linked to cellular differentiation and migration during embryonic development and 

pathological metastatic progression of cancers. Efforts to define pathway genes responsible for 

mediating TGFβ-induced EMT through transcriptomic profiling have yielded little information, 

suggesting the involvement of genes regulated at the translational level. Using two different 

EMT models, we have identified and elucidated a novel TGFβ translational regulatory pathway 

mediated by a 33-nt structural (‘BAT’; TGFbeta-activated translational) element in the 3’-UTR 

of two bonafide EMT inducer transcripts, disabled-2 (Dab2) and interleukin like inducer of EMT 

(ILEI). TGFβ activates a cascade in which protein kinase B/Akt phosphorylates heterogeneous 

ribonucleoprotein E1 (hnRNP E1) on Ser43, causing its release from the BAT element, 

culminating in translational activation of both Dab2 and ILEI mRNAs. Modulation of hnRNP E1 

expression, or its Ser43, not only alters the translational silencing of these transcripts, but also 

TGFβ-mediated EMT. Thus, translational activation of Dab2, ILEI, and possibly other 

transcripts, is suggestive of a post-transcriptional operon regulating TGFβ-induced EMT during 

development and metastatic progression of tumors. The autocrine response of cells to 

TGFβ-induced Akt activation and subsequent translational activation of EMT inducer 

transcripts may represent a novel mechanism through which the increased TGFβ expression in 

tumor cells contributes to cancer progression and provides avenues for novel anti-cancer 

therapeutic strategies.



CÁTIA IGREJA 

Multiple roles for CUP in translational repression 

Elisa Izaurralde, Cátia Igreja 

MPI for Developmental Biology - Tuenigen, Germany 

Drosophila CUP is an eIF4E-binding protein that acts as a translational repressor. Although its 

role is known to be crucial for diverse aspects of female germ-line development, the 

mechanism by which it represses translation is not fully understood. 

Here we report that CUP has multiple functions that induce and sustain translational repression 

in Drosophila S2 cells. 

We show that CUP can interact with eIF4G in an eIF4E-independent manner. This suggests 

that CUP may repress translation by interfering with the proper assembly of the eIF4F complex 

at the level of the cap structure and not solely by acting as a competitive inhibitor for the 

eIF4E-eIF4G interaction. 

On the other hand, when bound to a reporter transcript CUP can silence expression of this 

mRNA, also independently of eIF4E-binding. CUP-induced silencing promotes reporter 

deadenylation through recruitment of the CCR4-CAF1-NOT complex, but not degradation of 

the mRNA body. However, CUP-mediated translational repression can also occur in the 

absence of a poly (A) tail and in cells depleted of CAF1 or NOT1. This suggests that 

CUP-induced deadenylation is a consequence and not the cause of translational repression. 

We also observed that CUP coimmunoprecipitates with different decapping activators in an 

RNase A-independent manner and that these interactions displace these proteins from the 

P-bodies to the cytoplasm. Furthermore, CUP overexpression interferes with mRNA 

decapping. These results provide an explanation for why CUP-mediated deadenylation is not 

followed by decapping and subsequent mRNA degradation. 

Together these results indicate that CUP is able to repress translation in an eIF4E-independent 

manner and that translational repression is sustained by promoting transcript deadenylation. 

142

TOSHIFUMI INADA 

40S ribosome-bound RACK1 functions in nascent peptide-dependent 

translation arrest 

143 


Toshifumi Inada 1, Kazushige Kuroha 1, Lyudmila Dimitrova 1, Takehiko Itoh 2, Yuki Kato 2, 

Katsuhiko Shirashige 2 

1 Nagoya University, Japan 

2 Tokyo Institute of Technology, Japan 

Nascent peptide-dependent translation arrest plays a crucial role in quality control of eukaryotic 

gene expression. We have proposed that translation of the poly(A) sequence of nonstop mRNA 

results in translation arrest by the synthesis of poly-lysine and leads to co-translational protein 

degradation, thereby repressing the expression of the aberrant protein1,2. We have also 

reported that 12 consecutive basic amino-acid residues cause translation arrest followed by 

Not4p-mediated co-translational protein degradation by the proteasome3. Here we report the 

identification of a novel factor RACK1 that is involved in nascent peptide-dependent translation 

arrest. Mutational analysis of RACK1 revealed that the ribosome binding of RACK1 is crucial 

for translation arrest. Moreover, RACK1-dependent translation arrest stimulates an 

endonucleolytic cleavage of mRNA that is independent of Hbs1/Dom34. A truncated product 

of the cleavage, a nonstop mRNA without poly(A) tail, requires Dom34/Hbs1 for its translation, 

suggesting that the homolog of eRF1/eRF3 may be involved in termination codon-independent 

translation termination. 1Inada T. et al. (2005) <strong>EMBO</strong> J. 24:1584-1595. 2Ito-Harashima S. et al. 

(2007) Genes Dev. 21:519-524. 3Kuroha K. & Dimitrova L. et al. (2009) J. Biol. Chem. 

284:10343-10352.



JC JANG 

The Arabidopsis tandem zinc finger protein AtTZF1 traffics between the 

nucleus and cytoplasmic foci and affects development and hormone response 

Perry Blackshear 1, JC Jang 2, Marcelo Pomeranz 3, Pei-Chi Lin 3, Cyrus Hah 3, Shin Gene 

Kang 3 

1 NIEHS and Duke University, United States of America 

2 Ohio State University, United States of America 

3 Plant Biotechnology Center, Ohio State University, United States of America 

P-bodies (PBs) are specialized cytoplasmic foci where mRNA turnover and translational 

repression can take place. Stress granules (SGs) are distinct cytoplasmic foci, functionally 

related to PBs. The CCCH tandem zinc finger proteins (TZFs) play pivotal roles in gene 

expression, cell fate specification, and various developmental processes. Human TZF (hTTP) 

binds AU-rich elements at the 3’UTR, and recruit decapping, deadenylation, and exonucleolytic 

enzymes to PBs for mRNA turnover. It is not known if plant TZFs can bind RNA and localize to 

PBs or SGs. We have identified Arabidopsis AtTZF1 as a sugar sensitive gene in a microarray 

study. It is characterized by a TZF motif that is distinct from the human TZFs. Higher plants 

such as Arabidopsis and rice each have a gene family containing this unique TZF motif. Here 

we show that AtTZF1 can traffic between the nucleus and cytoplasmic foci. In plant cells, both 

AtTZF1 and hTTP co-localized with Arabidopsis AGO1, DCP2, and XRN4, the counterparts of 

mammalian PB/SG markers. AtTZF1-associated cytoplasmic foci are dynamic and 

tissue-specific. They can be induced by dark and wound stresses, and are preferentially 

present in actively growing tissues. Since AtTZF1 can bind both DNA and RNA in vitro, it raises 

the possibility that AtTZF1 is involved in transcriptional control in the nucleus, and RNA 

regulation in the cytoplasm. Consistent with this notion, we have found that AtTZF1 acts as a 

positive regulator of sugar and ABA (a stress hormone) response, and as a negative regulator 

of GA (a growth hormone) response. Gain-of-function plants are compact, late flowering, and 

cold/drought resistant. Microarray analysis reveals that ectopic expression of AtTZF1 mimics 

the effects of +ABA/-GA in gene expression. Notably, a gene network centered by a 

GA-inducible and ABA-repressible putative peptide hormone is severely repressed by the 

ectopic expression of AtTZF1. The role of AtTZF1 in mRNA turnover will be discussed. 

144

LUKASZ JAROSZYNSKI 

145 


Translational control of DDX3Y gene transcripts by miRNA binding in 3’ UTR 

Lukasz Jaroszynski 1, Christiane Jurek 1, Jutta Zimmer 1, Peter H Vogt 1 

1 University Women Hospital, Germany 

The DDX3Y gene, belonging to the DEAD-box RNA helicase gene family, is localised to the 

AZFa region in the long arm of the human Y chromosome. Its deletion was found frequently in 

infertile men with complete depletion of germ cells. Interestingly, although DDX3Y is commonly 

transcribed, translation is restricted to pre-meiotic male germ cells (Ditton et al., 2004). 

Recently we have found that the gene utilises three different polyadenylation signals (PAS), 

resulting in two short 3’ UTRs of 244 (PAS1) and 411 (PAS2) nts and one long (PAS3) of 2359 

nts. The longest transcript was expressed ubiquitously in all analysed human tissues while the 

shorter ones were found exclusively in testis. As 3’ UTR sequences are known targets for 

translational repressors (binding proteins and/or miRNAs), we wanted to know whether use of 

the testis specific PAS1/2 have a distinct role in the translational control of DDX3Y transcripts. 

For this purpose we have cloned different lengths of the DDX3Y 3’ UTR sequences after the 

luc2 gene in the pGL4.13 vector. Strong decrease in the reporter gene expression was 

observed only when the longest 3’ UTR was present in the construct. Interestingly, using the 

TargetScan programme for mapping miRNA binding sites between PAS2 and PAS3 we 

identified a number of miRNAs with a broad expression in all human tissues analysed. Since 

we wanted to know whether any of them is responsible for the observed Luc-reporter 

repression we destroyed their seed sites in the 3ÚTR and analysed the mutation effect by 

subsequent luciferase assays. Release of translational repression was observed after mutation 

of the miRNA 181 seed site. Quantitative evaluation, however, points to the possibility of more 

functional miRNA targets between PAS2 and PAS3. We, therefore, like to conclude that 

miRNAs expression in non-testis human tissues indeed contribute to the translational 

repression of DDX3Y transcripts with long 3ÚTRs



JACEK JEMIELITY 

Tetraphosphate mRNA Cap Analogues with high affinity for eIF4E and 

increased stability toward decapping enzymes 

Jacek Jemielity, Anna Rydzik, Malwina Strenkowska, Joanna Kowalska, Joanna Zuberek, 

Maciej Lukaszewicz, Zbigniew Darzynkiewicz Edward Darzynkiewicz 

University of Warsaw, Poland 

In vitro synthesized capped mRNAs have recently emerged as a promising alternative in 

therapeutic gene delivery. One of the limitations in their use is relatively poor stability in vivo. An 

intrinsic feature of all eukaryotic mRNAs, contributing to their stability, is m7G(5')ppp(5')N cap 

structure present at their 5' end. Previously we have shown that mRNAs possessing caps 

site-specifically modified with either methylenebis(phosphonate) or phosphorothioate moiety in 

the triphosphate bridge are resistant to Dcp2 deacapping enzyme and due to that have 

elongated half-lives in vivo [1,2]. We also showed that mRNA translational efficiency can by 

increased by improving its affinity to eIF4E translation factor e.g. by extending the cap 

5',5'-bridge to tetraphosphate [3]. Another motivation for designing enzymatically stable cap 

analogues with high affinity to eIF4E is their potential use as inhibitors of translation in vivo, 

what is important in designing anti-tumor drugs. 

We synthesized a series of mRNA cap analogues including ARCA [3] containing either 

methylenebis(phosphonate) or phosphorothioate modification in the tetraphosphate bridge. 

Fluorescence quenching experiments revealed that all of these compounds have significantly 

higher KAS for eIF4E than naturally occurring cap (up to 30-fold). Moreover, analogues 

modified with phosphorothioate moiety in non-bridging delta position as well as those modified 

with methylene group in gamma-delta bridging position of tetraphosphate are resistant 

towards degradation by human Decapping Scavenger (DcpS) as determined by an 

HPLC-based assay. We proved also that all tetraphosphate analogues are strong inhibitors of 

translation in RRL system as well as are capable of being incorporated into mRNA by T7 RNA 

polymerase and are efficiently translated in vitro. 

[1] Grudzien E. et al., J. Biol. Chem. 281, 1857-1867 (2006) 

[2] Grudzien-Nogalska E. et al., RNA 13, 1745-1755 (2007) 

[3] Jemielity J. et al. RNA 9,1108-1122 (2003) 

146

MARTIN JINEK 

147 


Biochemical and structural studies of the microRNA-mediated translational 

repression 

Marc Fabian 1, Nahum Sonenberg 1, Martin Jinek 2, Jennifer Doudna 2 

1 McGill University, Montreal, Canada 

2 University of California, Berkeley, United States of America 

MicroRNAs (miRNAs) are endogenous non-coding RNA molecules that post-transcriptionally 

regulate eukaryotic gene expression. Metazoan miRNAs assemble with Argonaute (Ago) 

proteins to elicit translational repression and/or decay of target mRNAs harbouring partially 

complementary sequences in their 3' untranslated regions. The mechanism of translational 

repression by metazoan miRNAs remains elusive. Recent data obtained from cell-free systems 

suggest that miRNAs inhibit translational initiation by interfering with the function of the cap 

recognition complex eIF4F. Proteins of the GW182 family interact with Ago proteins and are 

required for translational repression and mRNA decay elicited by microRNAs, as well as for 

localization of the miRISC complex in P-bodies. It has been shown recently that the 

glycine-tryptophan rich (GW) motifs of GW182 directly interact with the PIWI domain of Ago 

proteins. We have confirmed the hAgo2-GW182 interaction in vitro using recombinant proteins 

and show that dominant negative fragments of human GW182 protein TNRC6C efficiently 

inhibit miRNA-mediated translational repression and deadenylation in a cell-free translation 

system. This indicates that both miRNA-mediated translational repression and deadenylation 

are dependent on the interaction between Ago and GW182 proteins, and suggests that the 

Ago-GW182 complex is the effector of microRNA silencing. Our current experiments are aimed 

at testing whether GW182 family proteins are bona fide translational inhibitors and probing for 

their interactions with components of the translational machinery.



CATHERINE JOPLING 

Regulation of translation by microRNA-122 binding to the hepatitis C virus 5' 

untranslated region 

University of Nottingham, United Kingdom 

MicroRNA-122 (miR-122) is a liver-specific microRNA that interacts with the 5’ untranslated 

region (UTR) of hepatitis C virus (HCV) RNA, and exerts a positive effect on viral RNA 

abundance. This effect is mediated by direct binding of miR-122 to two adjacent seed match 

sequences that are separated by a short, conserved, spacer region. miR-122 binding to both 

sites is essential for HCV RNA levels to be maintained, and the integrity of the spacer region is 

also important. We have tested the effects of miR-122 on luciferase RNA bearing the HCV 5’ 

and 3’ UTRs, and observe a miR-122-dependent stimulation of translation. In contrast to the 

effects of miR-122 on HCV RNA abundance in the context of the full genome, we see an 

additive effect of the two miR-122 binding sites, rather than an absolute requirement for both 

sites. Analysis of different variants of the reporter RNA indicated that this translational 

stimulation is dependent on the RNA being uncapped, and that substitution of the HCV internal 

ribosome entry site (IRES) with that of encephalomyocarditis virus (EMCV) results in a 

significant reduction in translation activation. This suggests that microRNAs can positively 

regulate translation via 5’ UTR binding sites, but this regulation is dependent on the 

mechanism of translation initation. miR-122-dependent stimulation of translation of luciferase 

driven by the HCV 5’ UTR was shown by Henke et al, who also observed a miR-122-mediated 

increase in HCV translation at early timepoints (Henke et al, <strong>EMBO</strong> J (2008) 27:3300). We did 

not observe a significant effect of miR-122 on HCV translation by two experimental methods at 

later times, and concluded that the microRNA is likely to function at the level of viral replication. 

The extent to which miR-122 exerts its effects via regulation of HCV translation is therefore not 

yet clear. The implications of miR-122 binding during the HCV life cycle will be discussed. 

148

MARKO JOVANOVIC 

149 


A Quantitative Targeted Proteomics Approach to Identify microRNA Targets in 

C. elegans 

Paola Picotti 1, Vinzenz Lange 1, Ruedi Aebersold 1, Benjamin Hurschler 2, Xavier Ding 2, 

Helge Grosshans 2, Cherie Blenkiron 3, Eric Miska 3, Marko Jovanovic 4, Lukas Reiter 4, Erica 

Bogan 4, Manuel Weiss 4, Sabine Schrimpf 4, Michael Hengartner 4 

1 ETH Zurich, Switzerland 

2 FMI Basel, Switzerland 

3 University of Cambridge, United Kingdom 

4 University of Zurich, Switzerland 

Computational prediction methods for the identification of microRNA (miRNA) target genes 

face considerable challenges; in fact, potential miRNA targets predicted by three common 

algorithms overlap only by 10 to 20% in the nematode Caenorhabditis elegans. Furthermore, 

transcript profiling, the most commonly used method for experimentally detecting miRNA 

targets, only detects effects of miRNAs at the transcriptional level. Here we present a 

large-scale targeted proteomics approach to identify potential miRNA targets in C. elegans. 

Using selected reaction monitoring (SRM), we screened more than hundred potential let-7 

targets, which were then mined to select potential miRNA target genes of biological 

significance. In addition, we demonstrated by independent experimental downstream analyses, 

such as genetic interaction, polysomal profiling and luciferase assays, that the candidate genes 

classified as regulated by let-7 due to our proteomic quantitations, are indeed strongly 

associated to let-7 function. We propose that this method can be applied to validate candidate 

lists generated by computational methods or by large-scale experiments. Moreover, the 

method presented here can be easily adapted to other organisms.



PANAGIOTA KAFASLA 

Mapping the orientation of PTB binding to picornavirus IRESs 

Richard Jackson, Panagiota Kafasla, Helen Lin, Tuija Poyry, Nina Morgner, Carol Robinson 


We have recently used a combination of tethered hydroxyl radical probing and mass 

spectrometry to determine the stoichiometry and orientation of PTB (polypyrimidine tract 

binding protein) binding to the encephalomyocarditis virus (EMCV) IRES: which RBD binds to 

which site in the IRES? The mapping showed that a single PTB binds the core IRES in a 

unique orientation, with RBDs 1 and 2 binding near the 3’-end, and RBDS 3 and 4 near the 

5’-end, which would place constraints on the three-dimensional conformational flexibility of the 

IRES1. We have now used a similar approach to map the binding sites of each RBD on the 

poliovirus IRES, where our results suggest a rather different orientation from that seen with the 

EMCV IRES, with RBDs 1 and 2 being the main docking sites of PTB onto this IRES. In 

a different approach, aiming to ascertain which RBD/IRES interactions are particularly critical 

for stimulation of picornavirus IRES activity, we have made point mutations in the actual 

RNA-binding surface of each of RBDs 2, 3 and 4 and a linker replacement of the entire RBD1. 

Mutation of the binding surface of RBD 2 abrogated stimulation of the EMCV IRES, without 

seriously perturbing the interaction of the other three RBDs with the IRES. Similarly, stimulation 

of poliovirus IRES activity was abolished by disruption of RBD 2 binding. On the other hand, 

replacement of RBD1 by a linker had a much smaller effect on EMCV IRES activity, strongly 

implying that it is the binding of RBD2 rather than RBD1 to the 3’-end of the core IRES that is 

critical for stimulation. Preliminary results from experiments with mutations in the binding 

surfaces of RBDs 3 and 4 suggest that the two picornavirus IRESs have different requirements 

for interaction with these two RBDs. 

1Kafasla et al., Mol. Cell, in press, 12 June 

150

ZHALA KARIM 

The EF4 (LepA) Effect on Reverse Translocation 

151 


Knud H. Nierhaus 1, Hanqing Liu 2, Barry S. Cooperman 3, Zhala Karim 4, Markus Pech 4 

1 Max-Planck-Institut für Molekulare Genetik, D-14195 Berlin, Germany 

2 Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, United 


3 Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, United 


4 Max Planck Institute für Molekulare Genetik, Germany 

The recently detected elongation factor 4 (EF4), found almost in all bacteria, mitochondria and 

chloroplasts, is similar structurally to the translocase EF-G: EF4 lacks the subdomain G’ and 

domain IV of EF-G’s five domains and containing, in addition, a unique C-terminal domain. In 

contrast to EF-G, EF4 promotes reverse translocation of tRNAs on the ribosome, allowing for 

correction of defective translocation that occurs extensively, when the intracellular ionic 

strength is increased. Such an event prevents an osmotic collapse of the cell under ionic 

stress. 

With growth competition method, we show now that a ΔEF4 E. coli strain is overgrown by the 

wild type under stress conditions such as high salt, low pH and low temperature. Under 

moderate conditions i.e pH ~ 7, EF4 is only present in small fraction in the cytosol but in large 

amounts in the membrane. Increased ionic strength in the wild type strain cause a relocation of 

EF4 from the membrane to the cytoplasm. 

The clear in vivo importance of EF4 has led us to elucidate the mechanism of EF4-catalyzed 

reverse translocation, using kinetics assays measuring changes in the fluorescence of 

proflavin-labeled tRNAs and in the reactivity toward puromycin of ribosome-bound 

peptidyl-tRNA, as well as EF4-dependent GTPase. The results permit formulation of a 

quantitative kinetic scheme for EF4-catalyzed reverse translocation that proceeds from 

post-translocation complex (POST) to pre-translocation complex (PRE) via two intermediates: 

i.e., POST → I2 → I1 →PRE. EF4 does not catalyze I1 to PRE conversion. Spectinomycin 

inhibits reverse translocation by selectively stabilizing I2. A putative replacement of EF4 bound 

to I1 with EF-G would allow the ribosome to correctly translocate the bound tRNAs.



NAVAZ KARIMIAN POUR 

Translational Control of ApoB mRNA: Insulin Modulation via Localization in 

Cytoplasmic P Bodies 

Navaz Karimian Pour, Khosrow Adeli 


Apolipoprotein B (ApoB) synthesis and secretion is known to be partially regulated at the 

translational level; however, the molecular mechanisms that govern translational control of 

apoB mRNA remains largely unknown. Since apoB mRNA has a long half-life of 16 hours, its 

availability for translation could be regulated by the rate of release from translationally-silenced 

mRNPs within cytoplasmic foci called Processing Bodies (PBs). We investigated the role of 

PBs in controlling the availability of translationally-competent apoB mRNAs upon insulin stimuli. 

We assessed the co-localization of apoB mRNA with PB markers under time course insulin 

treatments by using a strong interaction between the bacteriophage capsid protein MS2 and a 

sequence specific RNA stem-loops. Co-transfection of HepG2 cells with two plasmids, one 

encoding an MS2 protein fused to GFP and the other one transcribing to several MS2 

stem-loops followed by the 15% of apoB mRNA and its UTRs helped us to visualize apoB 

mRNA. We observed a statistically significant increase in the localization of apoB mRNA into 

PBs after 4h, 8h, and 16h insulin treatments by 72%, 80%, 89%, respectively, compared to 

the control. However, acute insulin treatment did not show any significant effect on the 

localization of apoB mRNA into PBs. As a control, we visualized beta globin mRNA. There was 

no significant change in beta globin mRNA movement towards PBs in the presence of insulin. 

Overall, our data suggest that insulin may silence apoB mRNA translation by localizing its 

mRNA into PBs and reducing translationally-competent mRNA pools. Potential cis-trans 

interactions between apoB mRNA and putative RNA binding proteins present in PBs may 

regulate apoB mRNA silencing and may play an important role in dysregulation of hepatic 

apoB synthesis and secretion, as commonly observed in insulin resistant states and Type 2 

diabetes. 

152

RAY KELLEHER 

The functional roles of microRNAs in the developing and adult brain 

Harvard Medical School, United States of America 

153 


Translational control plays important roles in synapse development, synaptic plasticity and 

learning and memory. MicroRNAs are small noncoding RNAs that repress translation by 

recruiting the RISC complex to complementary sequences in the 3’ UTRs of target mRNAs. 

MicroRNAs have been implicated in synapse development and neurodegeneration, but little is 

known about the underlying mechanisms. To investigate the functions of microRNA-mediated 

translational control in the developing and adult brains, we have conditionally inactivated each 

of the ribonucleases required for microRNA biogenesis, Drosha and Dicer, either in neural stem 

cells during neural development or in excitatory neurons of the postnatal forebrain. Inactivation 

of microRNA expression in the adult brain enhances cerebral protein synthesis and selectively 

alters protein synthesis-dependent synaptic plasticity. With increasing age, conditional 

knockout mice lacking microRNA expression in the adult brain develop marked hyperactivity 

and widespread apoptotic neuronal death. Neurodegeneration is accompanied by activation of 

the unfolded protein response and accumulation of autophagosomes, lysosomal inclusions 

and ubiquitinated protein aggregates. These findings demonstrate that loss of 

microRNA-dependent translational repression leads to global dysfunction of neuronal 

mechanisms for protein homeostasis and quality control. Inactivation of microRNAs in neural 

stem cells does not affect brain regionalization or patterning but causes a severe defect in 

neurogenesis characterized by ER stress and widespread apoptosis. Collectively, our findings 

define a crucial role for maintenance of normal protein homeostasis by microRNAs in neural 

development, synaptic plasticity and neuronal survival.



SOHAIL KHOSHNEVIS 

Biophysical studies of interactions of eIF3i within the eIF3 complex 

Ralf Ficner, Sohail Khoshnevis 

1 Institute of Microbiology and Genetics, Georg - August - University, Goettingen, Germany 

Initiation of translation in eukaryotes is an orchestrated event including several protein factors 

called translation initiation factors (eIFs). The GTP-bound eIF2 carries the initiator methionyl 

tRNA into the P-site of the 40S ribosome where together with other initiation factors they form 

43S pre-initiation complex. The largest of the initiation factors is eIF3, with thirteen subunits in 

human and five stoichiometric subunits in budding yeast. The yeast eIF3 is shown to interact 

with eIF2.GTP.tRNAmet, eIF5 and eIF1 to form a multifactor complex which can exist free of 

ribosome. All five subunits of yeast eIF3 have homologs in human, suggesting that they form a 

functional core, which is capable of doing all the basic functions of eIF3. eIF3i (also known as 

Tif34 in yeast), a 40 kDa component of eIF3, is shown by biochemical methods to interact with 

eIF3g and eIF3b. We have purified eIF3i and studied its interaction with other components of 

eIF3 using biophysical approaches. We could detect the strongest interaction of eIF3i with 

eIF3g, suggesting them to form a stable complex. We aim to combine this information with 

structural data to gain a better understanding of the eIF3 compsition. 

154

JON HALVOR KNUTSEN 

A possible link between cell growth and the cell cycle: eIF2 alpha 

phosphorylation 

Jon Halvor Knutsen 1, Tonje Tvegård 1, Béata Grallert 1, Erik Boye 1 

1 Institute for Cancer Research, Norway 

155 


We have shown that ultraviolet (UVC) irradiation of fission yeast cells in G1 phase induces a 

delay in S phase entry (Nilssen et al, 2003). More recently, we found that the cell cycle delay is 

accompanied by a general depression of translation and phosphorylation of the translation 

initiation factor eIF2α (Tvegard et al., 2007). Both the cell cycle delay and downregulation of 

translation are absolutely dependent on the Gcn2 kinase, which is known to phosphorylate 

eIF2α at Ser52 and thereby reduce global translation. However, the checkpoint is only partially 

dependent on eIF2α Ser52 phosphorylation, indicating that there must be more Gcn2 targets 

in fission yeast. 

We reasoned that other phosphorylation sites on eIF2α might be targets of Gcn2. Such sites 

are predicted based on motif search and multiple sequence alignment between S. pombe, S. 

cerevisiae, and Artemia sp. We have prepared protein extracts from cells grown under different 

conditions and analyzed eIF2α by SDS-PAGE. Consistent with the conclusions based on 

sequence analysis, we have observed a band shift of eIF2α that might be due to modification 

events other than Ser52 phosphorylation. Furthermore, a recent paper described the 

identification of novel phosphorylation sites in the C-terminal end of eIF2α in fission yeast 

(Wilson-Grady et al., 2008). 

We are currently investigating whether modification at these and possibly additional sites on 

eIF2α are involved in the translation depression and in the G1/S delay after UVC irradiation, as 

well as the role of Gcn2 in these processes.



ANASTASSIA KOMAROVA 

Measles virus ribonucleoprotein core and cellular translational machinery 

Anastassia Komarova 1, Mathilde Guerbois 1, Chantal Combredet 1, Yves Jacob 1, 

Pierre-Olivier Vidalain 1, Frederic Tangy 1 

1 Pasteur Institute, France 

In cells infected with animal viruses, the regulation of translation plays a pivotal role in both viral 

replication and gene expression. This is achieved via viral protein interactions with cellular 

partners that often lead to modification of translation initiation factors. We have previously 

shown that Rabies virus matrix protein interacts with h subunit of eIF3 and that this interaction 

leads to inhibition of translation of classical Kozak-like RNAs in vitro (Komarova et al, 2007). 

Recently it has been found that the nucleoprotein (N) of Measles virus (MV, another member of 

Mononegavirales) also interacts with eIF3h (Sato et al, 2007), showing that different viruses 

share the same cellular targets. In the present work, we tested whether N associates with 

translation machinery in infected cells. Vero cells infected with the Schwarz MV vaccine strain, 

or recombinant MV-GFP were subjected to sucrose gradient analysis. Ribosome profiles were 

analysed and the distribution of N and GFP proteins was studied. The N protein was found 

similarly distributed throughout the gradient, whereas GFP was found only in the light fractions. 

Such distribution could reflect the fact that N is the major component of the viral RNP. 

However, we confirmed a co-localization of N with translational machinery by 

immunofluorescence. These data suggest that N could be associated with ribosomes in 

infected cells. However, this cannot simply be explained by N interaction with h subunit of eIF3 

since this cellular factor was observed only in a limited number of fractions within the same 

gradient. To identify the cellular partners of N during the replication cycle, we used reverse 

genetics approach to introduce tagged N protein within MV genome. This allows us to perform 

virus-host protein complexes purification by modified tandem affinity chromatography and 

mass spectrometry analysis. 

156

JOANNA KOWALSKA 

Boranophosphate Analogs of mRNA 5' end 

157 


Joanna Kowalska, Maciej Lukaszewicz, Joanna Zuberek, Zbigniew Darzynkiewicz, Edward 

Darzynkiewicz, Jacek Jemielity 


The 5'-cap is a distinguishing feature of all eukaryotic cellular mRNAs. It consists of 

7-methylguanosine connected via 5',5′-triphosphate bridge to the first transcribed nucleotide. 

Recently, we have used several synthetic cap analogs modified within 5',5'-phosphate bridge 

to produce mRNAs chemically modified at their 5' ends. Some of these mRNAs proved to be 

resistant to Dcp2 decapping enzyme, which resulted in their elongated half-lives and higher 

translational efficiencies in vivo. The ability to produce more stable mRNAs with higher 

translational efficiency can be advantageous for biotechnology as well as medicinal 

applications exploiting the concept of RNA-based gene delivery. 

On the other hand, cap analogs resistant to Decapping Scavenger (DcpS) were found to be 

strong and stable inhibitors of cap dependent-translation in cell free system. 

Here, we report the synthesis and biological properties of a new class of enzymatically resistant 

cap analogs bearing boranophosphate moiety at different positions of the 5',5'-triphosphate 

bridge (BH3-analogs). All of the studied analogs bind with high affinity for translational factor 

eIF4E (up to ~4-fold higher compared to m7GpppG). Some of the new compounds represent 

potent and DcpS-resistant inhibitors of cap dependent-translation. Despite the fact that DcpS 

carries out the hydrolysis by the attack on the cap γ-phosphate and in our previous studies 

only analogs modified at the γ position were resistant to DcpS we have surprisingly found that 

boranophosphate analogs modified at the β-position are also resistant to DcpS. Finally, the 

BH3-analogs possessing ARCA modification (which assures incorporation into mRNA only in 

the correct orientation) are suitable as reagents for preparation of capped mRNAs. Initial 

studies revealed that mRNAs capped with BH3-ARCAs are translated more efficiently than 

those capped with unmodified ARCA.



GÜNTER KRAMER 

The function of the chaperone trigger factor in co-translational folding of 

proteins 

Annemarie Becker, Günter Kramer 

Zentrum für Molekulare Biologie, Universität Heidelberg, Germany 

An essential step of protein synthesis is the successful de novo folding of the newly 

synthesized polypeptides. In all three kingdoms of life, a network of molecular chaperones 

exists to ensure efficient protein folding. When emerging from the ribosomal exit tunnel, 

nascent polypeptide chains are welcomed by ribosome-associated chaperones, in bacteria 

represented by trigger factor (TF). TF interacts with most emerging polypeptide chains and has 

been shown to support the de novo folding of many in particular larger polypeptides. How TF 

exerts its effect on these polypeptides is yet not clear though. Experiments measuring the 

activities of the model substrates eukaryotic firefly luciferase and the tetrameric b-galactosidase 

in vivo and in vitro suggest that TF causes a delay in the folding process (Agashe et al, 2004). 

However, it is not known yet whether this is a general mechanism of TF function valid for 

different substrates and how this can be correlated to its chaperone function. 

In the present study, we investigated the effect of TF on the folding of various E. coli proteins in 

a coupled in vitro transcription/translation system by monitoring the kinetics of disulfide bond 

formation as an indicator of tertiary structure formation. Substrate proteins were selected 

based on their dependence on various chaperones for folding as well as variable structural 

features. Indeed, TF postpones the formation of some disulfide bonds during synthesis such as 

the wildtype disulfide bond in b-lactamase, while the formation of others is not affected. 

Furthermore, tethering a protein distant enough to the ribosome to allow complete folding 

nevertheless seems to constrain its folding abilities, suggesting that ribosome proximity can 

interfere with de novo folding of some newly synthesized polypeptides. 

Literature: 

Agashe et al. (2004). Cell 117(2): 199-209 

158

HEIKE KREBBER 

The yeast mRNA export factor Npl3p functions in translation initiation 

159 


Thomas Gross 1, Baierlein Claudia 1, Alexandra Hackmann 1, Nicole Forster 1, Heike Krebber 2 

1 IMT, Germany 

2 Philipps-Universität Marburg, Germany 

The shuttling serine/arginine (SR)-type shuttling mRNA binding protein Npl3p is well known for 

its function in mRNA export from the nucleus to the cytoplasm. Npl3p associates with the 

mRNA already during transcription. Upon maturation of the mRNA it recruits the export factor 

heterodimer Mex67p-Mtr2p (TAP-p15 or NXF1-NXT1 in higher eukaryotes) to the 

ribonucleoparticle (RNP), which mediates the contact to the nuclear pore complex during 

transit of the mRNP to the cytoplasm. Interestingly, while bulk of the shuttling mRNA binding 

proteins including Mex67p dissociate from the mRNP during translation, Npl3p and two other 

yeast SR-proteins, Gbp2p and Hrb1p, remain associated with the mRNA during translation. 

We have shown earlier, that a mutation in NPL3, npl3-27, has defects in dissociation from the 

mRNA during translation elongation, which then in turn leads to defects in translation. 

However, if Npl3p has an active role during translation, or if these defects result from an 

inhibition of the moving ribosomes remained unclear. Now we show that Npl3p has an active 

role in translation initiation. We have generated mutants that exhibit no mRNA export defects, 

but exclusively defects in translation and present the analyses of such strains. Thus, we have 

separated both functions of Npl3p from each other and show here the characterization of the 

novel function of Npl3p in translation initiation.



POLINA KRJUCHKOVA 

Stop codon recognition sites in eRF1 

Polina Krjuchkova, Elena Alkalaeva1, Ludmila Frolova 

Engelhardt Institute of Molecular Biology, RAS, Russian Federation 

The final stage of protein biosynthesis is not as well understood as the preceding stages of 

translation (initiation and elongation). It occurs when the termination factor eRF1 recognizes 

one of three stop codons – UAA, UAG, UGA. It stimulates the hydrolysis of the ester bond in 

peptidyl-tRNA and peptide release. 

Previous studies have shown that the N-domain of eRF1 has two critically important amino 

acid motifs: NIKS and YxCxxxF (positions 61-64 and 125-131 in human eRF1 respectively). It 

was suggested that they are involved in stop codon recognition. 

Our results confirm the importance of amino acids from YxCxxxF motif. We specifically altered 

the stop codon recognition pattern of human eRF1 by point mutagenesis of conserved and 

semi conserved amino acids in different regions of N-domain. We obtained more than 60 

mutant release factors and determined their functional activity in reconstituted eukaryotic 

translation system. 

According to our data we suggest the existence of two sterically divided stop codon 

recognition sites in eRF1. One site decodes UAA and UAG stop codons and another site 

recognizes UGA stop codon. Moreover, we identify the amino acids responsible for conversion 

of omnipotent eRF1 to uni- or bipotent release factor. Our results provide new insights into 

stop codon decoding mechanism in eukaryotes. 

160

ANNA KROPIWNICKA 

161 


Binding affinities of eIF4E and eIF(iso)4E from Arabidopsis thaliana for mRNA 

cap analogues 

Anna Kropiwnicka, Joanna Zuberek, Joanna Kowalska, Maciej Lukaszewicz, Jacek 

Jemielity, Janusz Stepinski, Edward Darzynkiewicz 


For majority of eukaryotic mRNAs protein biosynthesis starts with a key step in which 

m7GpppN cap structure at the 5`-end is specifically recognised by the eukaryotic translation 

initiation complex 4F (eIF4F) consisting of a cap binding factor eIF4E and a scaffold protein 

termed eIF4G. Although many organisms posses multiple eIF4E family members, plants are 

unique among other Eukaryotes because they have two canonical translation initiation factors 

4E: eIF4E and eIF(iso)4E. The second factor together with its counterpart eIF(iso)4G make up 

an isozyme form of eIF4F complex named eIF(iso)4F. The reason why plants use both eIF4F 

and eIF(iso)4F complexes in translation initiation remains elusive. The in vitro biochemical 

studies (1,2) suggest that these complexes can discriminate between mRNA differing in their 

secondary structure or having internal initiation sites. However, up to date no biophysical 

studies that would compare eIF4E and eIF(iso)4E in their ability to bind mRNA 5`-end have 

been performed. Hence, we decided to determine binding affinities of eIF4E and eIF(iso)4E 

from Arabidopsis thaliana for a series of synthetic cap analogues by means of fluorescence 

titration method. We found out that eIF4E binds m7GTP and m7GpppG 14-fold and 3-fold 

weaker respectively, than its murine homologue, but 5-fold and 10-fold stronger than 

eIF(iso)4E. Therefore, two important questions arise: how eIF(iso)4E can compete with eIF4E 

for 5` mRNA terminus in plant cells and why plants need a second, functional in translation 

initiation, cap binding factor (eIF(iso)4E) as they already posses one (eIF4E) which has higher 

affinity for cap. 

1. Gallie DR & Browning KS (2001) J Biol Chem. 276(40):36951-60 

2. Gallie DR (2001) J Virol. 75(24):12141-52



DOROTA KUBACKA 

Investigating the cap-binding ability of the ovary-specific Xenopus eIF4E1b 

Nicola Minshall 1, Nancy Standart 1, Dorota Kubacka 2, Joanna Zuberek 2, Janusz Stepinski 2, 

Jacek Jemielity 2, Edward Darzynkiewicz 2 

1 Department of Biochemistry, University of Cambridge, United Kingdom 

2 Division of Biophysics, University of Warsaw, Poland 

Eukaryotic initiation factor 4E, named eIF4E1b, is a close homologue of the canonical class I 

eIF4E, (eIF4E1a; 71% identity). As a component of the CPEB complex along with the 

eIF4E-binding protein 4E-T(transporter), the Xp54 RNA helicase and other RNA-binding 

proteins, eIF4E1b is supposed to take a part in silencing translation during oocyte maturation 

and early stages of embryogenesis. In its primary structure occur all residues required to bind 

the cap (W56/W102/W166/E103/R112/R157/K162) but eIF4E1b in oocyte lysates only 

interacts weakly with m7GTP compared to eIF4E1a. Interestingly, several additional 

cap-proximal residues which may impact on cap-binding are found to systematically differ 

between vertebrate eIF4E1a and 1b proteins (1). 

Our experiments focus on understanding the difference in cap-binding between these two 

proteins and on the identification of the features of this process using the fluorescence titration 

method with recombinant proteins. eIF4E1b, which has a positively charged N-terminal region, 

requires a specialised protocol involving protein purification from inclusion bodies, using 6 M 

GdnHCl, separating it from nucleic acids and other particles, followed by an FPLC purification 

step. The instability problem in fluorescence measurements for this isoform is another 

challenge. The results from ongoing studies will be presented. 

1. Minshall, N., Reiter, M.-H., Weil, D., Standart, N., 2007 CPEB interacts with an 

ovary-specific eIF4E and 4E-T in early Xenopus oocytes. J. Biol. Chem. vol. 282 (52), 37389 

162

SVEN LAMMICH 

163 


The Expression of the alpha-secretase ADAM10 is regulated via its 5'UTR 

Claudia Prinzen 1, Falk Fahrenholz 1, Sven Lammich 2, Dominik Büll 3, Sonja Zilow 3, Frits 

Kamp 3, Brigitte Nuscher 3, Ann-Katrin Ludwig 3, Christian Haass 3 

1 Johannes Gutenberg Universität, Mainz, Germany 

2 Ludwig Maximilians Universität München, Germany 

3 Ludwig Maximilians Universität, München, Germany 

Alzheimer’s disease is the most common form of dementia worldwide. It is characterized by 

intracellular neurofibrillary tangles and extracellular amyloid plaques. Amyloid plaques consist 

mainly of the hydrophobic amyloid β peptide (Aβ), which is liberated upon endoproteolytic 

processing of the amyloid precursor protein (APP) by consecutive cleavages of β-secretase 

and γ-secretase. In contrast, cleavage of APP by α-secretase prevents the formation of Aβ. 

ADAM10 (a disintegrin and metalloprotease) was shown to be the best candidate for 

α-secretase in vivo. ADAM10 has a 5’ untranslated region (5’UTR), which belongs to the class 

of long 5’UTRs which were demonstrated to regulate translation of mRNAs from many 

regulatory genes. It is 444 bp long, has a GC-content of 70% and contains 2 upstream open 

reading frames. Here we show that the 5’UTR of ADAM10 regulates the rate of ADAM10 

translation. In the absence of the 5’UTR we observed a significant increase of ADAM10 protein 

levels in HEK293 cells. mRNA levels were not changed. Moreover, we could show that the 

5’UTR of ADAM10 inhibits the translation of luciferase in a coupled in vitro transcription and 

translation assay. We analyzed the effect of several deletion mutants in order to determine 

regions within the 5’UTR which influence the translation efficiency of ADAM10. Successive 

deletion of the first part of the ADAM10 5’UTR resulted in a significant increase in ADAM10 

protein expression, arguing that this part of the 5’UTR contains inhibitory elements. We provide 

evidence that a 30 nucleotide long G-rich stretch within the first part of the ADAM10 5'UTR is 

able to form an extremely stable intramolecular G-quadruplex secondary structure which 

contributes to the inhibitory effect of the 5’UTR on the translation of ADAM10.



MARLON LAWRENCE 

The Extended Loops of Ribosomal Proteins L4 and L22 are Not Essential for 

Ribosome Function, Cell Survival, or Peptide-Mediated Translational Arrest 

Marlon Lawrence, Lasse Lindahl, Janice Zengel 

University of Maryland, Baltimore County, United States of America 

Ribosomes are the universal organelles responsible for polymerizing amino acids into proteins. 

Ribosome-mediated peptide synthesis takes place within the peptidyl-transferase center of the 

large subunit of the ribosome. Once synthesized, nascent peptides traverse the ribosome exit 

tunnel before reaching the extraribosomal environment. Several nascent peptides have been 

shown to interact with the exit tunnel to stall translation elongation at specific sites within their 

peptide chain. The exit tunnel is primarily lined by rRNA, but the extended loops of ribosomal 

proteins L4 and L22 contribute to the lining, forming the narrowest portion of the tunnel. This 

constriction has also been shown to be important for the action of and resistance to 

macrolide-lincomycin-streptogamin (MLS) antibiotics. Several mutations localized near the 

constriction of the tunnel have been shown to interfere with nascent peptide-mediated 

pausing. Previous studies of MLS antibiotics led to the proposal that the extended loops of L4 

and L22 provide a gating mechanism by which the ribosome exit tunnel is narrowed or 

widened. The ability of these two proteins to adjust tunnel width has been suggested to play 

important roles in both antibiotic resistance and in regulating the passage of newly formed 

peptides through the ribosome exit tunnel. Through mutation and the use of translational 

reporters we have obtained results which show that, contrary to the proposed model, the 

extended loops of these two proteins are not required for ribosomal function, cell survival, or 

several cases of peptide-mediated translational arrest. We also present work showing that 

different classes of peptides known to stall translation respond differentially to mutations in L4 

and L22. 

164

MICHAEL LEICHTER 

165 


A potential role for the survival of motorneuron complex and methylosome in 

the assembly of selenoprotein mRNAs 

Michael Leichter, Laurence Wurth, Alain Krol, Christine Allmang 

Université de Strasbourg, CNRS, France 

Selenoprotein synthesis requires co-translational recoding of in-frame UGA codons. In 

eukaryotes, this process involves the assembly of RNA-protein (RNP) complexes to specific 

stem-loops located in the 3’UTR of selenoprotein mRNAs, called Selenocysteine Insertion 

Sequences (SECIS). 

Essential in this process is the SECIS binding protein 2 (SBP2) that binds the SECIS RNA and 

recruits translation and assembly factors to the mRNP. Our laboratory recently demonstrated 

that proper folding of SBP2 and selenoprotein mRNP assembly relied on a conserved 

machinery linked to the Hsp90 protein chaperone. The same folding machinery is required for 

the assembly of several other essential RNPs, such as small nucleolar RNPs, telomerase RNP 

and U4 small nuclear RNP. 

Here we show that SBP2 interacts with additional RNP assembly factors. Affinity purification of 

endogenous SBP2 from HeLa cell extracts followed by mass spectrometry analysis identified 

the MEP50 and PRMT5 argine-methyltransferase proteins as potential targets of SBP2. Both 

proteins are components of the methylosome, a complex which functions together with the 

survival of motorneuron (SMN) complex to assemble Sm as well as Sm like proteins on small 

nuclear RNAs. 

Immunoprecipitation experiments combined with yeast two-hybrid interaction tests allowed us 

to validate the in vivo association of SBP2 with both methylosome components and the SMN 

complex, in particular a subset of associated factors (gemins). Finally, pull down assays with 

E.coli expressed recombinant proteins demonstrated that SMN and the methylosome MEP50 

and pICln components can directly interact with SBP2. 

Together our results suggest that SMN and the methylosome complex participate in 

selenoprotein mRNP assembly, further supporting our discovery that the assembly of 

selenoprotein mRNPs exhibits even more striking similarities with that of sn(o)RNPs than initially 

anticipated.



RACHEL LERNER 

SLIP1 Plays a Role in Transport, Translation, and Degradation of Histone 

mRNA 

Rachel Lerner, Adele Ricciardi, Michael Slevin, Shawn Lyons, William Marzluff 

University of North Carolina- Chapel Hill, United States of America 

Histone mRNAs, unlike all other eukaryotic mRNAs, end in a conserved stem-loop rather than 

the canonical poly(A) tail. The stem-loop is bound by stem-loop binding protein (SLBP), which 

is required for histone mRNA processing, translation, and degradation. SLIP1, 

SLBP-interacting protein 1, was identified in a yeast-two-hybrid screen using SLBP as bait and 

binds to the region of SLBP required for translation. SLIP1 enhances the translation of reporter 

mRNAs ending in a histone stem-loop and is required for optimal levels of histone protein 

synthesis in vivo. Using RNAi we demonstrated that SLIP1, unlike SLBP, is required for cell 

viability. Since this data suggests that SLIP1 performs vital functions in addition to its role in 

histone mRNA translation, we performed a yeast-two-hybrid screen using SLIP1 as bait to gain 

further insight into the cellular functions of SLIP1. 

We identified SLIP1 binding partners with roles in RNA metabolism, including: 3’hExo, involved 

in mRNA processing; DEAD-box RNA helicase Dbp5, functioning in mRNA export; and eIF3g, 

a translation factor. We validated these interactions using GST pulldowns. Dbp5 is an mRNA 

export factor that has recently been shown to also have a role in mRNA translation termination 

(Gross T et al, 2007). Immunoprecipitation of endogenous SLIP1 co-IPs endogenous Dbp5, 

confirming that this interaction occurs in vivo. Current studies using dominant-negative Dbp5 

will determine the role of the SLIP1-Dbp5 interaction in histone mRNA export and translation. 

Histone mRNAs are rapidly degraded when DNA synthesis is inhibited. The first step in 

degradation is the addition of an oligo(U) tail to the 3’ end of the mRNA, which binds the 

Lsm1-7 complex. SLIP1 is associated with histone mRNA after addition of the oligo(U) tail via 

an interaction with Lsm1. Collectively these data suggest that SLIP1 is part of the histone 

mRNP starting in the nucleus and remains associated throughout the lifetime of the mRNA. 

166

ALAIN LESCURE 

Homozygous mutations in the SEPN1 gene affecting insertion of 

selenocysteine and causing rigid spine muscular dystrophy 

167 


Mathieu Rederstorff 1, Alain Krol 1, Pascale Richard 2, Ana Ferreiro 3, Pascale Guicheney 3, 

Valérie Allamand 3, Alain Lescure 4 


2 Institut de Myologie - INSERM, France 

3 Institut de Myologie - Paris, France 

4 Strasbourg University - CNRS, France 

Mutations in the SEPN1 gene encoding the selenium containing protein SelN cause a group of 

neuromuscular disease. Of particular interest are two mutations affecting the RNA cis 

sequences required for selenocysteine insertion. Recent studies have implicated mutations 

affecting both cis- and trans-acting factors of the selenocysteine insertion machinery in several 

human disorders. Analysis of related specific mutations in the SEPN1 gene provided valuable 

information about this peculiar translation mechanism. In the first case, the UGA selenocysteine 

codon was converted to a UAA stop codon, leading to premature termination of translation. 

The second concerns a single homozygous point mutation in the SEPN1 3’UTR SECIS 

element. This single-nucleotide modification was sufficient to abolish the binding of the SECIS 

recognition factor SBP2, a central component for selenocysteine translation, thereby 

preventing redefinition of the UGA selenocysteine and leading to a premature stop of 

translation. Interestingly, both mutations not only affected the synthesis of the protein, but also 

resulted in a drastic reduction of the SEPN1 mRNA levels. These observations demonstrated 

that the recognition of the selenocysteine codon as a premature stop induces destabilization of 

the SEPN1 transcript. Therefore, the selenocysteine insertion machinery appears to have a 

dual role: promoting the insertion of a selenocysteine residue into the polypeptide chain and 

protecting the selenoprotein encoding mRNAs against degradation by the mRNA 

quality-control machinery. In addition, our experiments provide interesting evidence that 

premature termination of translation due to nonsense mutations is amenable to correction, in 

the context of the specialized selenoprotein synthesis mechanism.



KIN-MEI LEUNG 

Live imaging of ß-actin mRNA transport in retinal axons and growth cones 

Kin-Mei Leung, Antonio Schmandke, Simon Bullock, Christine Holt 


Retinal ganglion cell axons navigate from the retina to the optic tectum. Growth cones (GC), 

the motile structures at the tip of growing axons, lead the way and make directional decisions 

based on encounters with different guidance cues. Guidance cues can elicit rapid local protein 

synthesis (PS) in GCs and direct their growth. Previous studies have shown that the 

chemoattractant, netrin-1, stimulates β-actin PS in GCs in 5min and a gradient of netrin-1 

induces polarized β-actin PS to the near-side of the GC. This asymmetry is generated at least 

in part by polarized transport of Vg1RBP (ZBP1 homolog), an RNA-binding protein known to 

interact with β-actin mRNA. Here we have focused on the dynamics of β-actin mRNA in 

growing axons and have investigated the effects of netrin-1 on mRNA transport. 

Fluorescently-labeled β-actin mRNA was transcribed by incorporation of Cy3-UTP and the 

mRNA was electroporated into embryonic eyes. Axon cultures were made from fluorescent 

retinas and used for time-lapse visualization of the mRNA in living neurons. We observed 

brightly labeled granules of β-actin mRNA that moved bi-directionally along the axons with 

60% moving in the anterograde direction at speeds of up to 180μm/min. The average 

anterograde speed is 52% faster than the retrograde speed in the axon compartment, but in 

the GC compartment the rates are similar in both directions. The anterograde transport of 

β-actin mRNA requires microtubules because nocodazole treatment dampened the transport. 

Bath addition of netrin-1 increased anterograde β-actin mRNA transport by 45% after 4min 

and induced granule trafficking from the GC central domain into the periphery. The results 

show that β-actin mRNA is rapidly transported into the GC and that netrin-1 causes a burst in 

the rate of trafficking. The findings are consistent with a role for β-actin mRNA trafficking as an 

underlying mechanism for cue-induced polarized β-actin PS. 

168

ACHIM LEUTZ 

Physiological relevance of the C/EBPbeta uORF 

169 


Cornelis Calkhoven 1, Achim Leutz 2, Valerie Begay 2, Jeske Smink 2, Klaus Wethmar 2 

1 Leibniz Institute for Age Research - Fritz Lipmann Institute, Germany 

2 Max-Delbrueck-Center for Molecular Medicine, Germany 

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) regulates 

proliferation and differentiation in many mammalian cell types. A single intronless C/EBPbeta 

transcript gives rise to short (LIP) and long (LAP*, LAP) protein isoforms, due to alternative 

translation initiation that is controlled by a small upstream open reading frame (uORF). We have 

examined the physiological relevance of alternative translation initiation by generating two 

murine mutant “knockin” (KI) strains that replace the endogenous C/EBPbeta gene and that 

may produce only the short LIP C/EBPbeta isoform or that lack the uORF and therefore fail to 

switch between isoforms. Both mouse strains have multiple phenotypes and details will be 

presented at the meeting. Our results show that uORF mediated C/EBPbeta isoform regulation 

plays an important role in metabolism, homeostasis and regeneration.



NOA LIBERMAN 

DAP5 - A Translation Initiation Factor that mediates Cap-Independent 

Translation 

Noa Liberman, Adi Kimchi 


DAP5/p97 (Death Associated Protein 5) is a member of the eIF4G family. The homology of 

DAP5/p97 to eIF4G is largely confined to the middle domain which corresponds to the eIF4A 

and eIF3 binding regions. The significant difference between the proteins is that DAP5/p97 

lacks the eIF4E binding site required for the interaction with the cap structure. This 

characteristic of the protein led us to the hypothesis that DAP5 mediates the cap-independent 

mode of translation initiation. Indeed we have shown that DAP5/p97 promotes IRES translation 

of the pro-survival proteins Bcl-2 and CDK1 and that knock-down of DAP5 leads to M-phase 

specific apoptotic cell death in HeLa cells. It was also shown in our lab that DAP5 is processed 

by caspases upon apoptotic stimulus, giving rise to a C-terminally truncated 86 kDa isoform. 

This isoform positively regulates IRES-driven translation of various mRNAs such as c-Myc, 

Apaf-1, XIAP and c-IAP1/HIAP2 including the IRES of DAP5 itself. In an effort to understand 

the differences in function and mode of action of DAP5/p97, DAP5/p86 and eIF4G our 

research has proceeded in several directions. This includes the search for new targets by 

analyzing the DAP5 knock down cells, the identification of DAP5 interacting proteins and 

resolving the crystal structure of DAP5 protein and its various domains. Understanding the 

mode of action of DAP5 and its partners may reveal the mechanism of IRES utilization and 

function in the life cycle of a cell, in developmental processes and in human diseases. 

170

CHIEN-LING LIN 

The Nuclear Experience of CPEB: Implications for RNA Processing and 

Translational Control 

University of Massachusetts Medical School, United States of America 

171 


CPEB is a sequence-specific RNA binding protein that promotes polyadenylation-induced 

translation in early development, during cell cycle progression and cellular senescence, and 

following neuronal synapse stimulation. It controls polyadenylation and translation through 

other effecter molecules, most notably the poly(A) polymerase Gld2, the deadenylating enzyme 

PARN, and the eIF4E-binding protein maskin. Here, we report that CPEB shuttles between the 

nucleus and cytoplasm and that its export occurs via the CRM1 dependent pathway. In the 

nucleus of Xenopus oocytes, CPEB is associated with lampbrush chromosomes and several 

proteins involved in nuclear RNA processing. CPEB also interacts with maskin in the nucleus 

as well as CPE-containing mRNAs. While the CPE does not regulate mRNA export, it does 

influence the degree to which mRNAs are translationally repressed in the cytoplasm. We 

propose that in Xenopus oocytes, association of the CPEB complex with mRNA in the nucleus 

ensures tight translational repression upon export to the cytoplasm. We also screened wild 

type and CPEB knockout mouse embryo fibroblasts (MEFs) for changes in alternative splicing 

using microarrays. We could identify a pre-mRNA, collagen 9a1 that underwent exon skipping 

in the absence of CPEB. Thus, at least in murine cells, CPEB is also involved in alternative RNA 

splicing.



JING-YI LIN 

Mechanism of negative regulation by Far upstream element binding protein 2 

interacts with enterovirus 71 internal ribosomal entry site 

Jing-Yi Lin, Shin-Ru Shih 

Chang Gung University, Taiwan 

The far upstream element binding protein 2 (FBP2) is also known as the KH-type splicing 

regulatory protein (KSRP). It was originally identified as a component of a protein complex that 

assembles on an intronic c-src neuronal-specific splicing enhancer, and as an important 

adenosine-uridine element binding protein (ARE-BP) that interacts with several AREs. 

Biotinylated RNA-affinity chromatography and proteomic approaches were employed to 

identify FBP2 as an internal ribosomal entry site (IRES) trans-acting factor (ITAF) for enterovirus 

71 (EV71). The interactions of FBP2 with EV71 IRES were confirmed by competition assay and 

by mapping the association sites in both viral IRES and FBP2 protein. During EV71 infection, 

FBP2 was enriched in cytoplasm where viral replication occurs, whereas FBP2 was localized in 

the nucleus in mock-infected cells. The synthesis of viral proteins increased in 

FBP2-knockdown cells that were infected by EV71. Various amounts of recombinant PTB 

protein, a positive ITAF of picornavirus, were added to the in vitro binding assay and the results 

showed that FBP2 outcompeted PTB for IRES binding. Results of this study suggest that 

FBP2 is a novel ITAF that interacts with EV71 IRES and negatively regulates viral translation. 

172

ZHAORU LIN 

Probing the Mysteries of Ribosomal Frameshifting using Antisense 

Oligonucleotides 

Zhaoru Lin 1, Ian Brierley 1, John Flanagan 2, Robert Gilbert 3 

1 Department of Pathology, University of Cambridge, United Kingdom 

2 STRUBI, University of Oxford, United Kingdom 


173 


Programmed ribosomal frameshifting is a translation recoding event in which the ribosome 

slips one nucleotide in the 3' (−1) direction, allowing the production of two proteins from a 

single mRNA at a defined ratio. Frameshifting requires at least two cis-acting mRNA signals; a 

heptameric slippery sequence and a downstream stimulatory mRNA secondary structure. 

Recent work has revealed that under certain circumstances, antisense oligonucleotides (AONs) 

can substitute for a stimulatory RNA stem-loop or pseudoknot. These AONs thus act in trans 

to stimulate frameshifting when bound to the mRNA downstream of the slippery sequence. By 

replacing the mRNA secondary structure with AONs, the various elements of frameshifting can 

be observed independently. 

Here we demonstrate that 2-O-methyl (2OMe) and Morpholino (MO) AONs are able to 

stimulate −1 frameshifting on a U-rich slippery sequence and to levels comparable to those 

seen with a coronavirus pseudoknot stimulatory RNA. Interestingly, the 2OMe AONs were also 

able to stimulate +1 frameshifting on the same slippery sequence. This may be correlated to 

the strong translational pause induced by the 2OMe AON in comparison to the MO AON. 

Further investigation revealed that the directionality (+ or −) and the magnitude of frameshifting 

is influenced both by the nature of the slippery sequence and the distance between the 

slippery sequence and stimulatory structure (spacer). Similar to stem-loop and pseudoknot 

mediated frameshifting, it seems likely that the AONs promote frameshifting by compromising 

the ribosome-associated helicase activity, but the 5' edge of the bound AON needs to be 

closer to the ribosome to exert its effect. We have prepared cryo-EM reconstructions of 2OMe 

AON-stalled eukaryotic ribosomes that may help to pinpoint the stage in elongation at which 

−1 or +1 frameshifting occurs.



WEIZHI LIU 

Structural Insights into Parasite eIF4E Dual Binding Specificity for Monomethyl 

and Trimethylguanosine mRNA Caps 

Anna Niedzwiecka 1, Weizhi Liu 2, David Jones 2, Craig McFarland 2, Jeffrey Kieft 2, Rui Zhao 2, 

Richard Davis 2, Janusz Stepinksi 3, Marzena Jankowska-Anyszka 3, Ryszard Stolarski 3, 

Edward Darzynkiewicz 3 

1 Polish Academy of Sciences, Poland 

2 University of Colorado School of Medicine, United States of America 


The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, and through its 

interaction with the scaffold protein eIF4G, recruits the ribosome to the mRNA for translation 

initiation. Parasitic nematodes and flatworms have mRNAs with the typical 

monomethylguanosine (m7G) as well as a trimethylguanosine (m2,2,7G) cap. The m2,2,7G cap 

is derived from spliced leader trans-splicing. We have characterized eIF4E with dual binding 

specificity for both caps from two parasitic helminths (Ascaris and Schistosoma) using 

quantitative fluorescent titration, ITC, NMR, and crystallography. We have determined the first 

crystal structure of eIF4E with dual binding specificity for m7G and m2,2,7G caps. The overall 

structure of these atypical eIF4Es is similar to other eIF4E structures. Parasite eIF4E proteins 

bind monomethyl cap in a manner similar to single specificity eIF4E forms. NMR chemical shift 

perturbation, crystallography and ITC analyses for eIF4E with m7G or m2,2,7G indicates that 

the mechanism of cap-binding differs in the two complexes and provides new insight into 

binding of the m2,2,7G cap. Furthermore, our data suggest a path for the RNA from eIF4E. 

Overall, these studies provide novel insights into how parasite eIF4E can bind TMG cap 

(compared to mammalian eIF4E which has very low affinity for TMG cap). Our studies may 

enable future development of potential drugs against a variety of trans-splicing parasitic worms 

infecting upwards of 2 billion people. 

174

MARCELO LOPEZ-LASTRA 

175 


The 5’UTR of the MMTV mRNA exhibits cap-independent translation initiation 

Felipe E. Rodriguez 1, J. Pablo Huidobro-Toro 1, Marcelo Lopez-Lastra 2, 

Maricarmen Vallejos 2, Pablo Ramdohr 2, Fernando Valiente-Echeverría 2, Karla Tapia 2 

1 Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad 

Católica de Chile, Chile 

2 Laboratorio de Virología Molecular, Facultad de Medicina, Pontificia Universidad Católica 

de Chile, Chile 

The 5'UTR of the full length mRNA derived from the infectious, complete mouse mammary 

tumor virus (MMTV) genome was cloned into a dual luciferase reporter construct containing an 

upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In 

nuclease treated rabbit reticulocyte lysate, the MMTV 5'UTR was capable of driving translation 

of the second cistron. Translational activity from the MMTV 5'UTR was resistant to 

cap-analogs and to cleavage of eIF4G by FMDV L protease. IRES activity was also 

demonstrated in the Xenopus laevis oocyte model system by microinjection of capped and 

polyadenylated bicistronic RNAs harboring the MMTV-5'UTR. Finally, transfection assays 

showed that the MMTV-IRES exhibits cell type dependent translational activity suggesting a 

requirement for as yet unidentified cellular factors for its optimal function. 

FONDECYT 1060655, FONDECYT 1090318, FONDAP 13980001 and MIFAB.



FABRIZIO LORENI 

PIM1 oncoprotein is destabilized by ribosomal stress and inhibits cell cycle 

progression 

Stefan Karlsson 1, Valentina Iadevaia 2, Sara Caldarola 2, Laura Biondini 2, Angelo Gismondi 2, 

Dianzani Irma 3, Fabrizio Loreni 4 

1 Department of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund 

University Hospital, Lund, Sweden, Sweden 

2 Dipartimento di Biologia, Università di Roma Tor Vergata, Roma, Italy, Italy 

3 Dipartimento di Scienze Mediche, Università del Piemonte Orientale, Novara, Italy 

4 University of Rome Tor Vergata, Italy 

PIM1 is an oncogenic kinase mainly expressed in hematopoietic cells. We have recently found 

that PIM1 interacts with ribosomal protein (RP)S19. A number of RP genes are mutated in 

Diamond-Blackfan anemia (DBA) patients that exhibit an excess of apoptosis of erythroid 

precursors. A current hypothesis on the mechanism of this disease is that the defect in 

ribosome synthesis (ribosomal stress) would activate a p53-dependent growth arrest in early 

hematopoiesis. To explore the possible implication of PIM1 in this mechanism we have 

analyzed its expression in cultured cell model systems. We found that depletion of RPS19 in 

K562 and TF-1 hematopoietic cell lines causes a drastic destabilization of PIM1. Consistent 

with this finding, lymphoblastoid cells from DBA patients and cells treated with drugs known to 

interfere with nucleolar functions showed a decrease of PIM1 levels. Moreover, we observed 

that the lower level of PIM1 in RPS19-deficient cells is associated to an increase of the cell 

cycle inhibitor p27Kip and to a block in cell proliferation even in the absence of p53. To 

demonstrate that the decrease of PIM1 could be the cause of the block of cell proliferation we 

transfected PIM1 in RPS19-deficient cells. Overexpression of PIM1 in these cells induces a 

recovery from the proliferation arrest caused by RPS19 deficiency. All these data suggest that 

PIM1 could play a role in the alteration of growth and apoptosis observed in hematopoietic 

cells from DBA patients. 

176

EUGENIE LUFT 

177 


Translational Control of Inducible Nitric Oxide Synthase by Arginine Availability 

and Arginase in vitro and in vivo 

Eugenie Luft 1, Andrea Debus 1, Ulrike Schleicher 1, Christian Bogdan 1, Till König 2 

1 University Clinic of Erlangen, Germany 

2 University Clinic of Freiburg, Germany 

Inducible NO synthase (iNOS) and its generation of NO from L-arginine are subject to 

transcriptional as well as posttranscriptional control by cytokines. Using primary mouse 

peritoneal macrophages we investigated by which mechanism interleukin (IL)-13 affects the 

expression of iNOS protein. For induction of iNOS the macrophages were stimulated with 

interferon (IFN)-gamma plus lipopolysaccharide (LPS). Preteatment with IL-13 downregulated 

the expression of iNOS protein, whereas the level of iNOS mRNA remained unaltered. The 

expression of iNOS protein was completely restored by the addition of arginine during the 

stimulation phase or the presence of an arginase inhibitor during the pretreatment phase. 

Suppression of NO production and iNOS protein, but not of iNOS mRNA, was also seen, when 

IL-13 was replaced by purified arginase or when the macrophages were stimulated with 

IFN-gamma/LPS in arginine-free medium. Arginine-deficiency specifically impaired the de novo 

synthesis of iNOS, but did not affect the production of other macrophage products or the 

overall protein synthesis. Taken together, these data suggest that the expression of iNOS is 

regulated translationally by arginine availability. Ongoing studies revealed that the translational 

control of iNOS protein by arginase also takes place in vivo in cutaneous leishmaniasis. In order 

to elucidate the mechanism of this posttranscriptional regulation we investigated whether 

arginine-deficient macrophages follow the canonical integrated stress response that in other 

cell types depleted of amino acids was characterized by the accumulation of phosphorylated 

eukaryotic elongation factor-2alpha. Unexpectedly, resting as well as stimulated inflammatory 

macrophages constitutively exhibited high levels of phosphorylated eIF2alpha, which were not 

further increased upon L-arginine starvation. Alternative modes of translational repression of 

iNOS need to be considered.



MACIEJ LUKASZEWICZ 

Phosphorothioate analogs of m7GTP are enzymatically stable inhibitors of 

cap-dependent translation 


M. Lukaszewicz, J. Kowalska, J. Zuberek,M. Ziemniak, E. Darzynkiewicz, J. Jemielity 

Division of Biophysics, University of Warsaw, 02-089Warsaw, Poland 

Eukaryotic mRNAs bear at their 5' ends a specific cap structure- m7G(5')ppp(5')N–a 

7-methylguanosine attached by 5'–5' triphosphate bridge to the first transcribed nucleoside. 

This structure is involved in numerous events in mRNA metabolism, including intracellular 

transport, translation and degradation. During initiation of translation the cap is specifcally 

recognized by the eukaryotic initiation factor eIF4E. eIF4E protein plays the important role in 

translational control of gene expression. It is also known that eIF4E is overexpressed in many 

types of tumor cells. It was demonstrated that targeting eIF4E may inhibit the growth of tumor 

cells and induce apoptosis. 

In this study we aimed for cap analogs that are potent inhibitors of cap-dependent translation 

and are stable in a cellular enviroment. We report properties of new potent inhibitors of 

translation, where two phosphorothioate analogs of m7GTP– diastereoisomers m7GTPαS(D1) 

and (D2)– showed notably high affinity to eIF4E (KAS values 3- and 1.5-fold higher than for 

m7GTP, respectively). The more potent of diastereoisomers, m7GTPαS(D1), inhibited 

cap-dependent translation in rabbit reticulocyte lysate ~8-fold and ~15-fold more efficiently 

than m7GTP and m7GpppG, respectively. Strikingly, both analogs fully retained inhibitory 

properties after incubation in RRL, whereas inhibition by unmodifed ones was distincly 

affected. Stability of tested analogs in RRL appears to correlate with their resistance to human 

DcpS enzyme. 

m7GTPαS(D1) and (D2), and other cap analogs, that appears to be stable in a 

translationally-competent lysate derived from reticulocyte cells, are potent compounds to test 

them as translational inhibitors in living cells. 

178

RADOSLAW LUKOSZEK 

179 


tRNA levels vary in Arabidopsis thaliana cells: Consequences for protein 

biosynthesis 

University of Potsdam, Germany 

The rate-limiting step of translation is accessibility of charged tRNAs. Each cell contains set of 

tRNA necessary for its needs. Arabidopsis thaliana nuclear genome possesses 309 unique 

tRNA coding genes to satisfy the 61 sense codons, however not much is known about their 

expression profiles. This excess of isoacceptors (i.e., more than one specie that pair to the 

same codon) are not all expressed at the same time. We use various techniques, including 

qRT-PCR, HPLC, deep-sequencing, to quantitatively determine the composition of the tRNA 

isoacceptors in cell culture, young and senescent leaves of Arabidopsis thaliana. Aim is based 

on the tRNA concentration to predict the rate of translation on a proteome-wide scale for cells 

comprising various tissues. Furthermore, the modifications of the tRNA nucleosides are 

analyzed in order to determine their influence on translation and plant viability. Using pull-down 

assays we also determine the specificity of the A. thaliana elongation factor towards various 

isoacceptor families (same amino acid, same anticodon) or isodecoders (same amino acid, 

different anticodon).



NANDINI MANICKAM 

Genetic analysis of translational accuracy 

Nandini Manickam, Nabanita Nag, Philip Farabaugh 

University of Maryland, Baltimore County, United States of America 

Every cell’s survival depends on the fidelity of its translational machinery. In spite of 

sophisticated proofreading mechanisms, errors do occur in translation at a frequency of 10-3– 

10-4 per codon. Errors in translation can occur due to incorrect aminoacylation of a particular 

tRNA by its synthetase or selection of an incorrect tRNA by the ribosome. Aminoacylation has 

been shown to be a very accurate process, so the decoding process contributes more 

towards the translational errors. There are three types of decoding errors: processivity errors, 

misreading errors and frameshift errors. Of these three errors, I’m interested in estimating the 

misreading error frequency. Misreading is the misincorporation of an amino acid. Emily Kramer 

in our laboratory used a dual luciferase reporter system to study the misreading of all possible 

near cognate and non cognate codons of Lysine 529, an essential amino acid in the active site 

of the firefly luciferase. I’m using a similar, lacZ based reporter system to characterize the 

misreading of codons by several tRNAs in E. coli. Hence, my goal is to generate a reporter 

system for translational accuracy. Queuosine is a highly conserved modification in position 34 

of the tRNA. It is a hypermodified guanosine nucleoside present in Tyr, His, Asn and Asp 

tRNAs in position 34 (wobble) which are four of the essential active site amino acids in 

β-galactosidase. Absence of Q modification leads to amber mutation suppression, which 

suggests that Q modification may play an important role in decoding for certain tRNAs. Q 

modification might play two kinds of role in maintaining the translational accuracy. It might 

stabilize the correct base pairing between codon and anticodon or might destabilize incorrect 

base pairing. Either of these two hypotheses predict a specific change in misreading with our 

reporter system. 

180

CHRISTOPHE MARIS 

181 


Structural investigation of IRES RNA stemloop H of EMCV virus in complex 

with PTB RRM1 

Christophe MARIS, Frédéric H.-T. ALLAIN 

ETH Zurich, Switzerland 

Encephalomyocarditis virus (EMCV) initiates protein synthesis by internal ribosome entry site 

(IRES) mediated translation. The 450 nt-long IRES located its 5’UTR mRNA binds most of 

initiation eukaryotic factors to recruit the 40S subunit1. To be fully functional, EMCV IRES 

mRNA requires some IRES Trans-Acting Factors (ITAFs). Two identical apical UCUUU 

pentaloops were found to bind the Polypyrimidine Tract Binding protein (PTB). The four RNA 

recognition Motif (RRM) domains2 of PTB have different specificity and affinity for CU motifs3. 

We have solved the structure of the stem-loop H of EMCV IRES in its free form and in 

complex with PTB RRM1. The apical pentaloop forms a structured loop containing a U-U 

mismatched base-pair. Upon RNA binding, PTB RRM1 unfolds most of the loop in order to 

specifically recognize the CUU motif. The loops 1 and 3 and the very conserved C and N 

termini of PTB RRM1 contribute to the interactions. Unexpectedly, RNA binding induces a 

C-terminal helix at the interface of the complex. Because of additional protein contacts to the 

RNA stem, the stem-loop has an affinity five-fold stronger to the stem-loop than the one to the 

single stranded RNA (Kd of 5 μM compared to 25 μM). 

These data suggests that the secondary structure of the RNA is critical for the recruitment of 

ITAFs to the EMCV IRES. 

1. Pisarev, A. V., Shirokikh, N. E. & Hellen, C. U., C R Biol 328, 589-605 (2005). 

2. Kolupaeva, V. G., Hellen, C. U. & Shatsky, I. N., Rna 2, 1199-212 (1996). 

3. Oberstrass, F. C. et al., Science 309, 2054-7 (2005).



ALINE MARNEF 

Pat1 and Pat2 proteins are RNA-binding proteins that repress translation in 

Xenopus laevis oocytes 

Aline Marnef, Nancy Standart 


Pat1 has recently been identified as a component of a large mRNP complex containing 

CPEB1, responsible for the repression of mRNAs in Xenopus laevis oocytes. Relatively little 

characterised, Pat1 proteins in yeast, Drosophila, C. elegans and human somatic cells have 

been shown to be components of processing bodies (P-bodies), distinct cytoplasmic foci that 

are the sites of translational repression and RNA decay. In yeast, Pat1p is an RNA-binding 

protein that plays roles in activation of decapping, translational repression and P-body 

formation2. Yeast and invertebrates posses only one Pat protein, whereas vertebrates have 

evolved two paralogues: Pat1 and Pat2. 

Unusually, no known motifs can be identified in Pat proteins, providing no hint of function. 

xPat1/2 are differentially expressed in oocytes and early embryos, with xPat2 expression 

essentially taking over that of the maternal xPat1, which is degraded upon meiotic maturation. 

Using the MS2-tethering assay, we showed that xPat1/2 were able to repress translation in 

oocytes, and are currently delineating the domain responsible for the repression. Using an in 

vitro assay, xPat1/2 were identified as RNA-binding proteins capable of binding U and G 

RNA-homopolymers. They appear to bind RNA in a distinct fashion with xPat2 mainly binding 

RNA via a central region of the protein. Further work will involve the NMR study of this novel 

RNA-binding domain. Lastly, to unravel other potential functions of xPat1/2, we are also 

focusing on their interacting partners in oocytes and embryos. 

1. Minshall et al., 2007, J. Biol. Chem., 282, 37389-37401. 

2 Pilkington and Parker, 2008, Mol Cell Biol., 28,1298-312. 

182

ENCARNA MARTINEZ-SALAS 

Identification of Gemin5 as a novel IRES transacting factor 

Encarna Martinez-Salas, David Pineiro, Almudena Pacheco, Jorge Ramajo 

Centro de Biologia Molecular, Spain 

183 


In eukaryotic cells translation initiation occurs through two alternative mechanisms, a 

cap-dependent operating in the majority of mRNAs, and a 5´ end-independent driven by 

internal ribosome entry site (IRES) elements acting in a specific subset of mRNAs. IRES 

elements recruit the translation machinery to an internal start codon through a mechanism 

involving the IRES structure and several trans-acting factors. With the aim to discover novel 

factors involved in IRES-dependent translation, we have performed a proteomic approach to 

identify host factors interacting with two viral IRES, the foot-and-mouth disease virus (FMDV) 

and hepatitis C virus (HCV). RNA-affinity chromatography purification followed of mass 

spectrometry analysis allowed the identification of host factors that specifically recognize the 

IRES RNA. Proteins interacting with unrelated RNAs used as negative controls were 

disregarded for posterior analysis. Proteins specifically interacting with the FMDV IRES include 

several eIFs and hnRNPs known to interact with this IRES element, thereby validating the 

results obtained in this approach. Gemin5, an RNA-binding protein of 170 kDa, was found to 

interact with both IRES using two independent approaches, riboproteomic analysis and 

immunoprecipitation of photocroslinked factors. Functional analysis performed in Gemin5 

shRNA-depleted cells or in in vitro translation reactions revealed an unanticipated role of 

Gemin5 in translation control, acting as a down-regulator of cap-dependent and IRES-driven 

translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of 

two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent 

protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also 

functions as a modulator of translation activity.



DENISA MATEUS 

Molecular reconstruction of a Candida genetic code alteration in 

Saccharomyces cerevisiae 

Denisa Mateus, Manuel Santos 

Universidade de Aveiro / CESAM, Portugal 

Various alterations to genetic code have been found in bacteria, mitochondria and cytoplasm 

of eukaryotes, invalidating the Frozen Accident theory of genetic code. In several species of 

Candida genus, leucine CUG codons are decoded as serine by the mutant tRNACAGSer, 

which is recognized by Seryl and Leucyl-tRNA synthetases being aminoacylated with both 

serine and leucine. This dual recognition indicates that it must contain identity elements for 

both aaRSs. In silico studies revealed this tRNA was created through an adenosine insertion in 

the middle position of the anticodon loop of a tRNACGASer gene. This mutation changed the 

tRNA anticodon from 5’-CGA-3’ (serine) to 5’-CAG-3’ (leucine), but didn’t change its identity, 

creating a new tRNACAGSer that mistranslated leucine CUG codons as serine. Two additional 

mutations, namely A37-G37 and U33-G33 occurred in this tRNA. We are reconstructing in vivo 

this evolutionary pathway by expressing wild type and the mutant versions of the C. albicans 

tRNACGASer gene in S. cerevisiae. We studied the effect of each mutation on tRNA stability, 

aminoacylation, decoding efficiency and growth rate of S. cerevisiae. Our results show that 

mutations introduced in the anticodon of tRNACGASer gene do not affect the recognition of 

this tRNA by SerRS. Unexpectedly, sharp decrease in levels of the mutant misreading tRNAs 

was observed by Northern blot. This reduced tRNA expression was linked to the misreading 

phenotype rather than to tRNA destabilization by the mutations introduced in the tRNA gene. 

Data suggests the toxicity of the original mutant tRNAs, which initiated CUG identity redefinition 

in Candida spp., may have been minimized by low tRNA abundance rather than decreased 

aminoacylation or decoding efficiency. We are investigating the molecular nature of the low 

abundance of the recombinant tRNAs expressed in S. cerevisiae. 

184

FABIENNE MAUXION 

Control of mRNA deadenylation by BTG/Tob factors 

Fabienne Mauxion 1, Sabine Dessaigne 2, Samira Aït-Abdellah 2, Bertrand Séraphin 2 

1 CGM-CNRS, France 

2 CNRS-FRE3144, France 

185 


The BTG/Tob family comprises a group of antiproliferative proteins, which are characterized by 

a conserved N-terminal domain named the APRO domain. Although proposed to affect 

transcription regulation, all the BTG/Tob factors interact directly with Caf1, a subunit of the 

main eukaryotic deadenylase. Accordingly, it was recently demonstrated that expression of 

two members of this protein family, Tob1 and BTG2, are general activators of mRNA 

deadenylation (Ezzeddine et al. 2007. Mol Cell Biol. 27:7791-801; Funakoshi et al. 2007. 

Genes Dev. 21:3135-48; Mauxion et al. 2008. <strong>EMBO</strong> J. 27:1039-48). The results suggest that 

Tob1 activates mRNA deadenylation by recruiting the Ccr4/Caf1 deadenylase to mRNAs via its 

interaction with the poly(A)-Binding-Protein PABPC1. However, BTG2, like BTG1, BTG3 and 

BTG4, is not known to interact with PABPC1, or other general components of mRNPs, and 

the mechanism by which BTG2 activates deadenylation remains unknown. As BTG2 has been 

described to interact with PRMT1, a Protein Arginine Methyltransferase, we speculated that 

arginine methylation events could be involved in BTG2 activation of deadenylation. However, 

our results do not support this hypothesis. Interestingly, a structure-function analysis of 

BTG/Tob factors reveals that the APRO domains of BTG2 and BTG1 are sufficient to activate 

deadenylation whereas Tob1’s APRO domain is not active. Experiments are underway to 

identify the specific characteristics of BTG1/BTG2’s APRO domains required for activation of 

mRNA deadenylation.



ROBERT MC MAHON 

The role of host cell signaling and eIF4F in reactivation of quiescent herpes 

simplex virus type 1 (HSV-1) 

National Institute for Cellular Biotechnology, Ireland 

HSV-1 is a widespread human pathogen that exists in two distinct states. During lytic infection 

the virus actively replicates and ultimately kills the host cell. However, after primary infection 

HSV-1 enters a non-productive state, referred to as latency in vivo or quiescence in vitro, 

which allows the virus to colonize its host for life. Periodic reactivation most commonly results 

in recurrent cold sores but also causes corneal blindness and encephalopathy. However, our 

understanding of the events that underlie reactivation remains limited, due largely to difficulties 

associated with modeling this state in vitro. We recently described an efficient tissue culture 

model of quiescent HSV-1 infection using wild-type virus that allows direct comparison of the 

processes involved in lytic replication and virus reactivation in the same cell type. Here, we 

show that in contrast to lytic infection, where previous reports have shown that ERK is 

suppressed and p38 regulates the activity of the eIF4E kinase, Mnk1, during reactivation ERK 

activity was transiently stimulated and inhibitors of either ERK or Mnk1 suppressed viral antigen 

accumulation and infectious virion production, while a p38 inhibitor was ineffective. Rapamycin, 

which blocks mTOR-mediated phosphorylation of p70S6K and the translational repressor 

4E-BP1, also potently inhibited reactivation. Finally, the eIF4E:eIF4G inhibitor 4EGi-1 

suppressed reactivation in a dose-dependent manner. Our results demonstrate that efficient 

reactivation of dormant HSV-1 from within an infected cell requires the activity of the translation 

initiation complex eIF4F as well as signaling through both mTOR and ERK-Mnk1 host protein 

kinase pathways. These findings highlight mechanistic similarities as well as significant 

differences in how lytic replication and reactivation of HSV-1 exploit host translational control 

pathways, in particular the use of opposing signal pathways to regulate Mnk1. 

186

CELINE MESTEL 

Overexpression of eIF4E regulates tumor cell invasion largely through 

translational control of ß1 integrin mRNA 

Celine Mestel, Robert Schneider 


187 


eIF4E is frequently overexpressed in tumors of the breast, colon, prostate, and lung, and 

expression correlates with disease progression, increased tissue invasion and metastasis. It 

has been suggested that overexpression of eIF4E contributes to malignancy by selectively 

increasing translation of mRNAs containing significant secondary structure in the their 5’ NCRs. 

A limited group of mRNAs encode key proteins involved in cellular growth, survival, 

angiogenesis and malignancy and contain long GC-rich highly structured 5’NCRs. The effect of 

excess eIF4E in tumor cells, however, is still not well understood, nor is the role of 4E-BP1. 

Here we demonstrate the surprising finding that overexpression of eIF4E acts primarily to 

increase breast cancer cell invasion by significantly increasing translation of a single mRNA: β1 

integrin. eIF4E overexpression at levels found in breast cancers (3-4 fold) results in a 10-fold 

increased level of tumor cell invasion. Gene array analysis of polysomal mRNA found increased 

translation of β1 integrin mRNA with physiological levels of eIF4E overexpression observed in 

breast cancers. We found that this increased β1 integrin expression was due to increased 

read-through of the complex GC rich 5’NCR of β1 integrin mRNA following eIF4E 

overexpression, the first time this has been described physiologically, and that this effect is 

blocked by co-overexpression of 4E-BP1. Co-overexpression of a mutant 4E-BP1 lacking the 

eIF4E binding region does not effect the increased β1 integrin expression seen with eIF4E 

overexpression. Thus, increased expression of eIF4E at typical human breast cancer levels 

mediates increased tumor cell invasion through selectively increased translation of the β1 

integrin mRNA. 4E-BP1 may be acting as a tumor suppressor or invasion suppressor by 

neutralizing the translational effects of excess eIF4E in the cell.



POHL MILON 

Kinetic mechanisms on mRNA selection by the ribosome 

Pohl Milon 1, Andrey L. Konevega 2, Marina Rodnina 3, Claudio Gualerzi 4 

1 Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany 

2 Max-Planck-Instute for Biophysical Chemistry, Department of Physical Biochemistry, 

Germany 

3 MPI for Biophysical Chemistry, Germany 

4 University of Camerino, Italy 

Regulation of gene expression at the translational level can account for differences of up to 

three orders of magnitude. At the first phase of translation initiation, mRNA and initiator 

fMet-tRNAfMet bind to the 30S subunit with the help of initiation factors (IFs) resulting in the 

30S initiation complex (30S IC). The second phase comprises the association of the 50S 

subunit with the 30S IC, GTP hydrolysis, and the dissociation of IFs; this phase is considered 

irreversible and results in the 70S initiation complex (70S IC). In the present work we analyzed 

the mechanism of mRNA selection by the ribosome by measuring the rate constants of 30S IC 

and 70S IC formation. Our rapid kinetic measurements combined with earlier data (Studer and 

Joseph, 2006) suggest that upon the 30S IC formation, mRNAs are primarily selected 

according to their cellular concentrations, the lack of a significant secondary structure at the 

translation initiation region (TIR), and the ability to form a stable 30S IC. In the 30S IC, the 

stabilities of the fMet-tRNAfMet and mRNA increase, while that of IF3 decreases reciprocally by 

over 2 orders of magnitude. The stability switch is abolished when non-canonical mRNAs are 

used (i.e. with a near-cognate initiation codon). Whenever a correct 30S IC is formed, the 50S 

subunit can rapidly join the complex, IFs dissociate and the first peptide bond can be formed. 

However, if a 30S IC harbors a non-canonical mRNA, a dramatic reduction of the rates of 

subunit joining, IF3 and IF1 dissociation and peptide bond formation is observed. These data 

indicate the existence of a second kinetic checkpoint of translation initiation which occurs upon 

formation of the 70S IC and senses the properties of the TIR of the mRNA. 

188

ERIC MISKA 

189 


LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing 

in Caenorhabditis elegans 

Eric Miska, Nic Lehrbach 


The let-7 microRNA (miRNA) is an ultraconserved regulator of stem cell differentiation and 

developmental timing, and a candidate tumour suppressor. In C. elegans and other animals 

the expression of let-7 is developmentally regulated, but the mechanisms underlying this 

regulation remain unknown. Lin28 is a conserved RNA-binding protein, which in mammals 

controls stem cell lineages and inhibits let-7 miRNA processing in vitro. However, the precise 

mechanism and in vivo significance of this activity are unclear. Here we show that LIN-28 and a 

poly(U) polymerase, PUP-2, regulate let-7 processing in C. elegans. We have developed a 

quantitative in vivo assay of let-7 function, which reveals post-transcriptional regulation of let-7 

during development. We demonstrate that lin-28 is necessary and sufficient to block let-7 

activity in vivo; LIN-28 directly binds let-7 pre-miRNA to prevent Dicer processing. We have 

also identified additional miRNAs that appear to be targeted by LIN-28 in this way. As a further 

step towards elucidating the mechanism of let-7 regulation, we have identified a poly(U) 

polymerase, PUP-2, which regulates the stability of let-7 pre-miRNA under LIN-28 control. We 

show that PUP-2 and LIN-28 interact directly, and that LIN-28 stimulates uridylation of let-7 

pre-miRNA by PUP-2 in vitro. In addition, we show that pup-2 contributes to regulation of a 

stem cell lineage in vivo. Our results demonstrate that LIN-28 and let-7 form an ancient 

regulatory switch, conserved from nematodes to human, and provide insight into the 

mechanism of LIN-28 action in vivo. Uridylation by a PUP-2 orthologue might regulate let-7 

and additional miRNAs in other species. Given the roles of Lin28 and let-7 in stem cell and 

cancer biology, we propose such poly(U) polymerases are potential therapeutic targets.



SARAH MOHAMMAD-QURESHI 

Characterisation of Phosphoresidues within the Catalytic Subcomplex of eIF2B 

Sarah Mohammad-Qureshi, Rebecca Tyler, Graham Pavitt 

The University of Manchester, United Kingdom 

eIF2B, an essential guanine nucleotide exchange factor (GEF), catalyses release of GDP from 

inactive eIF2, allowing formation of active eIF2▪GTP and continued rounds of translation 

initiation. During cellular stress, GEF activity is regulated and inhibited indirectly, by 

phosphorylation of eIF2 - by the eIF2B regulatory subcomplex (subunits α, β and δ). 

Direct regulation of eIF2B can occur by targeting the catalytic subcomplex (subunits γ and ε), 

such as phosphorylation of mammalian eIF2B. Our study characterises phosphoresidues in the 

yeast eIF2B catalytic subcomplex to identify their roles in regulating GEF activity. We identified 

phosphoresidues in yeast eIF2Bγ and ε by mass spectrometry and protein biochemistry. We 

mutated these and other residues to Alanine (non-phosphorylatable) or Glutamic acid/Aspartic 

acid (phospho-mimicking). Growth of mutant yeast under non-optimal conditions 

highlighted residues necessary for wt eIF2B function. eIF2B γ and ε mutants display 

phenotypic responses to rapamycin and 3AT and have altered growth at low temperature. The 

most detrimental effect was observed with an eIF2Bε mutation; this exhibits extreme growth 

retardation under all stresses tested, including an inability to overcome amino acid starvation 

via the general control pathway (Gcn- phenotype) suggesting an extra regulatory step in the 

starvation response. In addition, we have antibodies specific to phosphoresidues whose 

mutation gave observable phenotypes; these are being optimised to measure how 

phosphorylation of eIF2B relates to the response of yeast to sub-optimal growth conditions. By 

relating this to in vitro studies of mutants, we can speculate on novel pathways which directly 

control eIF2B activity. 

190

MARTIN MOKREJŠ 

Deciphering the transcriptome of all eIF4E class I, II and III genes from 

full-length cDNA, EST, HTC data from most organisms 

Martin Mokrejš, Martin Pospíšek 

Charles University, Faculty of Science, Czech Republic 

191 


The N7-methylguanosine cap moiety at the 5' mRNA end plays a key role in recognition and 

binding of the mRNA molecule by eukaryotic ribosome. The eukaryotic translation initiation 

factor eIF4E has the unique capability to recognize the cap structure and functions thus as a 

highly specific forceps picking up mRNAs for the cellular translation initiation machinery. In the 

past a survey of various eIF4E forms has been done and the results have shown that eIF4E 

proteins can be classified into three classes. Most importantly, members of class II and III were 

found only in certain taxonomic groups. Further to note, the three classes differ in their ability to 

bind the cap structure, eIF4G protein and their susceptibility to be tuned by 

(de)phosphorylation. 

With the accumulation of genomic and EST data for several other eukaryotic organisms it is 

now possible to vastly refine previous findings. We found that fungal eIF4E proteins of class I 

from Basidiomycetes differ significantly from that of Ascomycetes. We found additional species 

of fungi containing eIF4E of the class II in their genome. Our analysis is focused also on 

mammalian eIF4E proteins and we found additional forms of all eIF4E1, eIF4E2 and eIF4E3 

genes at the transcript level. We show that transcription of human chromosomes 4, 5 and 17 

results in 14 transcript variants of just eIF4E1. The scene gets more crowded with additional 

transcripts of eIF4E2 and 3 class representatives from chromosomes 2 and 3, respectively. 

The overall view of the transcriptome brings up several interesting questions about functions of 

each of the proteins. The up to date analysis covers fungi, protozoa, and animals while 

emphasizing several model organisms: Tribolium, Drosophila, honey bee, mouse and human. 

The analysis also emphasizes that several transcripts are not covered by NCBI RefSeq or 

organism-specific genome annotation projects despite their experimental evidence at RNA 

level.



FRANCESCA MORETTI 

Positional effects of microRNA-mediated translational regulation and their 

mechanistic basis 

Francesca Moretti, Rolf Thermann, Matthias W Hentze 

EMBL, Germany 

MicroRNAs are key regulators of gene expression in a wide range of biological processes. 

However, the exact mechanism(s) by which microRNAs mediate regulation continue to be 

intensively investigated. 

MicroRNAs were initially found to mediate translational regulation by binding to the 3’ 

untranslated region (3’UTR) of target mRNAs. However, the insertion of microRNA binding 

sites within the 5’UTR or the open reading frame (ORF) of reporter mRNAs can induce a 

silencing response, and effective microRNA binding sites have been discovered in ORFs and 

5’UTRs of endogenous mRNAs. Although highly informative, these studies do not provide 

detailed understanding of the molecular mechanisms underlying the observations made. 

Taking this into consideration, we have set out to systematically investigate how the position of 

microRNA binding sites in mRNA reporter constructs influences microRNA-mediated 

translational regulation. For this purpose, we have generated reporter constructs containing six 

Drosophila melanogaster miR-2 binding sites alternatively in the 5’ or 3’ UTRs or the ORF. The 

analysis of these reporters shows that specific miR-2-mediated translational responses are 

elicited independently of the position of the microRNA binding sites, both in an in vitro system 

consisting of D. melanogaster cell-free embryo extracts and in vivo in D. melanogaster 

Schneider cells. We observe that miR-2 triggers deadenylation of all the reporter constructs 

without concurrent mRNA destabilization. Finally, we show that miR-2 inhibits 80S ribosome 

assembly and induces the formation of pseudo-polysomes from both UTRs and from within 

the ORF. 

In summary, our work establishes a mechanistic basis for miR-2 function from the 5’UTR and 

the ORF in addition to the proposed 3’UTR mechanism. 

192

CHRISTINE MÜLLER 

193 


Nucleolar retention of a translational C/EBPalpha isoform stimulates rDNA 

transcription and cell growth 

Christine Müller, Sabrina Eichwald, Sandra Schreiber, Götz Hartleben, Cornelis Calkhoven 

Leibniz Institute for Age Research - Fritz Lipmann Institute, Germany 

The messenger RNA of the intronless CEBPA gene is translated into three protein isoforms 

through the usage of consecutive translation initiation sites. These translational isoforms have 

contrasting functions in the regulation of differentiation and proliferation due to the presence of 

different N-terminal sequences. Here we describe the function of an N-terminally extended 

protein isoform of C/EBPalpha that is translated from an alternative non-AUG initiation codon. 

We show that Extended-C/EBPalpha but not the other translational isoforms of C/EBPalpha is 

retained in the nucleoli. A basic amino acid motif within the N-terminus of Extended- 

C/EBPalpha is required for nucleolar retention and for interaction with nucleophosmin (NPM). In 

addition, Extended- C/EBPalpha interacts with UBF and stimulates rRNA synthesis, and its 

expression results in increased cell size. Furthermore, during differentiation of HL-60 cells 

endogenous expression of Extended-C/EBPalpha is lost concomitantly with nucleolar 

immunostaining of C/EBPalpha probably reflecting the reduced requirement for ribosome 

biogenesis in differentiated cells. Altogether, our results suggest that stimulation of rRNA 

synthesis is a novel function of C/EBPalpha adding to its role in the regulation of differentiation 

and proliferation. The fact that this novel function can be specifically attributed to one 

translational isoform further strengthens the importance of regulated translation of the 

C/EBPalpha mRNA.



VANDA MUNZAROVA 

Mutational analysis of the interaction between the N-terminal domain of eIF3a 

and the 5’ enhancer of uORF1 from the GCN4 mRNA leader that is critically 

required for efficient reinitiation 

Josef Panek 1, Vanda Munzarova 2, Istvan Danyi 2, Leos Valasek 2 

1 Laboratory of Bioinformatics, Institute of Microbiology AVCR, v.v.i., Prague, the Czech, 

Czech Republic 

2 Laboratory of Regulation of Gene Expression, Institute of Microbiology AVCR, v.v.i.,, Czech 

Republic 

Reinitiation is a gene-specific translational control mechanism that utilizes short uORFs to 

down- or up-regulate translation of many regulatory proteins. Ribosomes initiate at the first 

AUG codon, however, at the termination codon where the 60S subunit dissociates, the 40S 

subunit remains mRNA-bound, resumes scanning, and initiates again at a downstream start 

site. Perhaps the best studied example of such a mechanism is the intricate regulation of 

expression of yeast GCN4 whose mRNA leader contains four uORFs. The very first uORF1 

differs from the others in the presence of specific 5’ and 3’ adjacent enhancing elements that 

are critically required for efficient resumption of scanning. We recently showed that tahe 5’ 

enhancer functionally interacts with the N-terminal domain (NTD) of the a subunit of translation 

factor 3 (eIF3a), presumably as it emerges from the mRNA exit pore, and that this interaction is 

critically required to promote retention of post-termination 40S subunits on the mRNA. Here 

we present detailed mapping analysis of the eIF3a-resposive site within the 5’ sequences of 

uORF1, and vice versa, with help of computer modeling. We revealed two separate clusters 

within the first 180 amino acid residues of eIF3a, the mutations of which impair GCN4 

regulation. Similarly, three discernible nucleotide stretches were found in the 5’ sequences of 

uORF1 that together account for its highly stimulatory activity. Genetic analysis strongly 

suggests that only the most proximal site is directly engaged in interacting with eIF3a-NTD, 

whereas the other two act independently. Owing to the fact that the postulated interaction 

must necessarily be very weak to allow resumption of scanning, we have not been able to 

detect direct binding in vitro. To circumvent this obstacle, we have developed an in vivo 

RNA-immunoprecipitation assay with specialized constructs carrying either solitary uORF1 or 

uORF4 from the GCN4 mRNA leader, the data of which will also be presented. 

194

NICOLO' MUSNER 

195 


Aanalysis of the stress transducer, PERK, in sciatic nerves of the CMT 1B 

neuropathy mouse 

Nicolo' Musner, Maurizio D'Antonio, Desirèe Zambroni, M. Laura Feltri, Lawrence Wrabetz 

San Raffaele Scientific Institute - DIBIT, Italy 

Charcot-Marie-Tooth 1 disease is a common inherited neuropathy. This pathology is 

characterized by the loss of myelin sheath integrity in the Peripheral Nervous System (PNS) 

resulting in slowed nerve conduction velocity, hind limb muscular atrophy, and postural 

abnormalities. It is caused by mutations in a wide range of genes; one of them is Myelin Protein 

Zero (MPZ), which is expressed by Schwann cells, the myelin forming glia of the PNS. MPZ 

encodes the most abundant protein of peripheral myelin and is required to compact its 

structure allowing fast conduction. When MpzS63Δ, a mutant causing CMT1B in humans, is 

expressed in mouse with wild type alleles, it produces a demyelinating neuropathy that mimics 

the corresponding human disease. MpzS63Δ is correctly expressed and translated but it does 

not reach the myelin sheath being retained in the endoplasmic reticulum (ER). The ER 

accumulation of unfolded proteins is generally followed by induction of the Unfolded Protein 

Response (UPR), an adaptive mechanism aimed to relieve ER stress. S63Δ accumulation 

triggers a dose dependent, UPR with increased phosphorylation of eIF2alpha, Atf6 cleavage, 

Ire-1 induced Xbp-1 splicing and Chop induction. Genetic ablation of Chop restores motor 

capacity and ameliorates electrophysiological and morphological abnormalities in S63Δ mice, 

suggesting that the UPR is pathogenetic and maladaptive. Since Chop is downstream of the 

Perk/eIF2alpha pathway, we studied the effects of Perk ablation in normal and S63Δ mice. 

Preliminary behavioural, morphological and biochemical data from these mice suggest that 

Perk is maladaptive in S63Δ nerves.



ASTRID MUSNIER 

Developmental regulation of p70 S6 kinase by a G protein-coupled receptor 

dynamically modelized in primary cells 

Anne Poupon 1, Pascale Crépieux 1, Astrid Musnier 2, Domitille Heitzler 2, Thomas Boulo 2, 

Sophie Tesseraud 2, Guillaume Durand 2, Eric Reiter 2 


2 INRA, France 

Signalling networks which regulate the translational machinery have been poorly investigated in 

response to activation of G protein- coupled receptors (GPCR), when compared to tyrosine 

kinase growth factor receptors (TKR). Here, we analyzed the regulation of p70 S6 kinase 

(p70S6K) in response to the Follicle-stimulating Hormone Receptor (FSH-R) as a model GPCR, 

in primary rat Sertoli cells at two developmental stages. We report that p70S6K admits several 

active, differently phosphorylated isoforms in response to FSH, depending on the FSH-R ability 

to mediate reciprocal production of second messengers, cAMP and PIP3, according to the 

developmental stage. Importantly, insulin-mediated p70S6K activation was consistent with 

extensively reported data, whatever the cell stage. By using data fitting, a dynamic 

mathematical modelling of p70S6K regulation by FSH signalling was obtained, and led us to 

estimate the respective level of three phosphorylated isoforms of p70S6K, a feature currently 

inaccessible to experimentation. The outcomes on the recruitment of p70S6K to the 5'cap of 

mRNA have also been examined. Therefore, our work underscores that several 

phosphorylated isoforms of p70S6K can be active, as a function of the developmental stage of 

Sertoli cells (proliferating versus differentiating cells), and the type of receptor engaged (GPCR 

versus TKR). 

196

ISABEL NAARMANN 

197 


DDX6 is a novel regulator of reticulocyte 15-lipoxgenase mRNA translation 

Isabel Naarmann 1, Christiane Harnisch 1, Bodo Moritz 1, Nadine Flach 1, Henning Urlaub 2, 

Dirk H. Ostareck 3, Antje Ostareck-Lederer 3 

1 Martin-Luther-University Halle-Wittenberg, Germany 

2 Max-Planck-Institute for Biophysical Chemistry, Germany 

3 University Hospital, RWTH Aachen, Germany 

During their maturation erythroid precursor cells loose the capacity for mRNA synthesis due to 

extrusion of the nucleus. Therefore, the stability and translation of mRNAs coding for specific 

proteins which function in late stages of maturation is controlled tightly. Reticulocyte 

15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes in 

the peripheral blood. To prevent a premature synthesis and activation of r15-LOX, which would 

disturb energy metabolism, r15-LOX expression is temporally restricted. R15-LOX mRNA can 

be detected in erythroid precursor cells, but the r15-LOX protein is only expressed in mature 

reticulocytes. HnRNP K and hnRNP E1 bind to the differentiation control element (DICE) in the 

3’UTR of the mRNA and thereby inhibit the joining of the 60S ribosomal subunit to the 43S 

preinitiation complex (1). So far it is not well understood how the interaction between the 43S 

preinitiation complex and the hnRNP K- hnRNP E1- DICE complex is achieved. 

To get further insight into this mechanism of posttranscriptional control of gene expression we 

set up an inducible erythroid cell system based on human K562 cells. This system fully 

recapitulates the DICE dependent translational regulation (2). To identify so far unknown factors 

involved in DICE dependent translational control we employed GRNA chromatography in 

combination with hnRNP K immunoprecipitation. Specifically co-purified proteins were 

identified by mass spectrometry. The interaction of candidate proteins with hnRNP K was 

further analyzed by immunprecipitation and GST pulldown assays. With these methods DDX6 

was identified as DICE dependent interaction partner of hnRNP K. Employing RNAi technology 

we could show that DDX6 is an important regulator of translation of the endogeneous r15-LOX 

mRNA in K562 cells. 

1) Ostareck et al. (2001) Cell 104, 281-290 

2) Naarmann et al. (2008) JBC 283 18461-18472



AINAOUI NADERA 

FGF1 induction in myogenesis depends on IRES novel cross-talks with 

promoter and 3’UTR elements 

INSERM, France 

Fibroblast growth factor 1 (FGF1) is involved in muscle development and regeneration, and 

required for muscle fiber formation. The FGF1 gene structure is complex as it contains four 

tissue-specific promoters allowing, by a process of alternative splicing, synthesis of four 

transcripts with distinct leader regions. Two of them contain internal ribosome entry sites 

(IRESs), which are RNA elements allowing mRNA translation to occur in conditions of blockade 

of the classical cap-dependent mechanism. 

We have investigated the molecular mechanisms regulating FGF1 expression during myoblast 

differentiation. We show that FGF1 is induced in differentiating myoblasts and regenerating 

mouse muscle, and that such induction is both transcriptional and translational, involving 

specific and simultaneous activation of FGF1 promoter A and IRES A at day 2 of differentiation, 

when cap-dependent translation is down-regulated. Furthermore, IRES activation is drastically 

increased by the presence of a mRNA 3’UTR element. 

Strikingly, we show that transcriptional and translational inductions of FGF1 are molecularly 

coupled, as IRES-driven translation is clearly activated by a promoter cis-acting element. The 

mechanisms controlling these cross-talks of FGF1 IRES with promoter and 3’UTR have been 

addressed by the biacore leading edge technology coupled with mass spectrometry, in order 

to identify protein complexes bound to FGF1 promoter DNA, as well as to FGF1 IRES and 

3’UTR RNA during myoblast differentiation. Biacore technology turned out to be successful for 

protein recovery and identification. New data about identification of potential ITAFs regulating 

the FGF1 IRES activity will be presented. 

These data reveal a novel mechanism of regulation of IRES-dependent translation, involving 

both promoter and 3’UTR, which has dramatic consequences on a physiological event, that is, 

muscle development. 

198

JAGPREET NANDA 

199 


eIF1 controls multiple steps in start codon recognition during eukaryotic 

translation initiation 

Jagpreet Nanda 1, Julie Takacs 1, Jon Lorsch 1, Yuen - Nei Cheung 2, Martin-Marcos Pilar 2, 

Alan Hinnebusch 3, Adesh Saini 4 

1 Johns Hopkins School of Medicine, United States of America 

2 LGRD, National Institute of Child Health and Human Development, NIH, United States of 

America 

3 National Institutes of Health, United States of America 

4 NICHD, NIH, United States of America 

eIF1 plays a key role in start codon selection. It acts as a negative regulator of overall GTP 

hydrolysis by eIF2 by preventing Pi release at non-AUG codons. It also promotes an open form 

of the 40S subunit that is thought to be competent for scanning the mRNA. Once the start 

codon has been recognized, eIF1 is released from the pre-initiation complex (PIC), triggering Pi 

release from eIF2. Recent work from our lab has indicated that base-pairing between the start 

codon and the initiator tRNA anticodon induces a conformational change in the PIC from an 

open state to a stable, closed one. The observed rate of eIF2•GTP•Met-tRNAi ternary complex 

(TC) binding to the 40S subunit in vitro reflects the rate of conversion of the PIC into this stable 

state. We now show that, in addition to regulating Pi release, eIF1 plays a key role in controlling 

the rate of conversion from the open complex to the closed one. We demonstrate that the 

charge around the factor’s penultimate residue (G107) is important for regulating its functions. 

Mutations that increase the positive charge density reduce the observed rate of TC binding as 

well as the rate of eIF1 release. In contrast, decreased positive charge around position 107 

increases the rates of TC binding and eIF1 release. The strong correlation between the rates of 

eIF1 release and observed TC binding, coupled with our previous data indicating that the 

observed rate of TC binding reflects the rate of the open-closed transition, provide evidence 

that eIF1 release is intimately coupled to this conformational change. In addition, our data 

indicate that eIF5 promotes release of eIF1 from the PIC. We propose a model in which eIF1 

and the N-terminal domain of eIF5, which have very similar folds, compete for binding to the 

same site in the PIC. Release of eIF1 upon start codon recognition allows eIF5 to enter this 

site, inducing a rearrangement in the complex and allowing downstream events in initiation.



SAWSAN NAPTHINE 

Translation termination – reinitiation in murine norovirus 

Cambridge University, United Kingdom 

Expression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is 

believed to occur by the unusual mechanism of termination codon-dependent reinitiation of 

translation. In this process, following translation of an upstream open reading frame (ORF) and 

termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA 

and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent 

with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 

ORF (UAA) in the pentanucleotide UAAUG. Here, we confirm the use of this mechanism in VP2 

expression and define the mRNA sequence requirements. Termination-reinitiation is dependent 

upon 43nt of RNA immediately upstream of the UAAUG motif. Chemical and enzymatic 

probing revealed that the RNA in this region is not highly structured and includes an essential 

stretch of bases complementary to 18S rRNA helix 26. Thus, similar to other viruses that use 

this strategy, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S 

subunit to the mRNA following termination at the VP1 stop codon. Unexpectedly, 

termination-reinitiation in MNV was found to be relatively insensitive to the initiation inhibitor 

edeine, suggesting that accurate recognition of the VP2 ORF AUG is not a pre-requisite for 

efficient reinitiation of translation in this system. 

200

MARIE NAVEAU 

Role of e/aIF2 subunits in initiator tRNA binding 

Marie Naveau 1, Yves Mechulam 2, Emmanuelle Schmitt 2 

1 Ecole Polytechnique-CNRS, France 

2 Ecole Polytechnique-CNRS UMR7654, France 

201 


The eukaryotic or archaeal GTP-bound initiation factor e/aIF2 supplies the ribosome with 

Met-tRNAiMet. After pairing of the tRNA anticodon with the initiation codon, e/aIF2 leaves the 

initiation complex in a GDP bound form. e/aIF2 is a heterotrimeric protein (α, β and γ subunits). 

γ binds the two other subunits which do not interact together. The role of each subunit in GTP 

dependent-tRNA binding was studied by determining the binding constants to the 

Met-tRNAiMet of monomers (α, β, γ), of heterodimers (αγ, βγ) and of the heterotrimer. In the 

case of archaeal aIF2, the isolated γ subunit is able to bind initiator tRNA. However, α is 

required for efficient binding, whereas β only plays a minor role. These results contrast with the 

major role of β and the minor role of α in tRNA binding proposed for eukaryotic eIF2. Therefore, 

a "eukaryotic behavior" would be opposed to an "archaeal behavior". To identify which subunit 

is responsible for the eukaryotic behavior, the binding constants of the initiator tRNA were 

determined for chimeric complexes formed by yeast αY and βY bound to archaeal γA. A 

eukaryotic behaviour of the chimeric factors was observed. Crystals of the αYγA chimera were 

obtained. Despite a poor resolution of 4.3 angströms, the structure shows that αY is bound to 

γA in a manner similar to that observed in the archaeal complex. Then, the absence of αY’s 

effect on tRNA binding is not due to abnormal linking to γA. In addition, we demonstrate that 

the eukaryotic behavior is independent of the structural extensions of βY, specific of eukaryotic 

β. Thus, the eukaryotic behavior of eIF2 relies on features within the sequence of the 

conserved domains of α and β.



ANNA NIEDZWIECKA 

Molecular mechanism of the mRNA 5’ cap binding by poly(A)-specific 3’ 

ribonuclease (PARN) 

Anna Niedzwiecka 1, Remigiusz Worch 1, Marzena Jankowska-Anyszka 2, Edward 

Darzynkiewicz 2, Per Nilsson 3, Niklas Henriksson 3, Anders Virtanen 3 

1 Institute of Physics, Polish Academy of Sciences, Poland 


3 Uppsala University, Sweden 

The mRNA 5’ cap structure is a primary anchor to the translation initiation factor eIF4E and 

protects the mRNA against 5’ exonucleolytic degradation. The cap is also involved in the 

mRNA 3’processing, since it stimulates the activity of poly(A)-specific exoribonuclease (PARN). 

PARN plays a key role in deadenylation in early development, is involved in nonsense-mediated 

mRNA decay, and also in regulation of cytoplasmic polyadenylation. There is a competition for 

the access to the cap between both proteins in the cell. 

The goal of our study was to elucidate how the 3’-specific deadenylase PARN binds the 5’ 

cap and to find structural requirements for this affinity. We have determined equilibrium 

dissociation constants for the cap binding to PARN, its point mutants and the RNA 

Recognition Motif (RRM) by means of intrinsic protein fluorescence quenching. The results 

show that PARN binds the cap with micromolar affinity and the RRM domain is responsible for 

this interaction. Contrary to all known RRMs, the PARN RRM does not exploit the canonical 

aromatic residue (His449) at the centre of the beta-sheet but Trp475 at the outer surface. 

Kinetic parameters describing PARN interaction with the cap have been estimated by surface 

plasmon resonance. They reveal that the complex formation is a slow, diffusionally controlled 

process. However, due to a very low kinetic dissociation constant, the interaction is stable. 

This behaviour results from that the PARN-cap intermolecular contacts are dominated by 

hydrophobic and van der Waals interactions with a negligible contribution from coulombic 

interactions. The results can explain why the cap is responsible for the processivity of the 

mRNA deadenylation by PARN. The kinetic data together with the equilibrium dissociation 

constants for cap analogues provide an insight into the molecular mechanism of the cap 

recognition by PARN and enable a comparison with eIF4E. 

202

MICHAEL NIEPMANN 

203 


Influence of the Hepatitis C Virus 3´-untranslated region on IRES-dependent 

and IRES-independent translation initiation 

Michael Niepmann 1, Christiane Bung 1, Zanda Bochkaeva 2, Ilya Terenin 2, Ivan Shatsky 2 

1 Justus-Liebig-University Giessen, Germany 

2 Moscow State University, Russian Federation 

The hepatitis C virus (HCV), a member of the Flaviviridae, has a positive-strand RNA-genome 

with a polyprotein open reading frame flanked by 5´- and 3´- untranslated regions (UTRs). The 

5´-UTR contains the internal ribosome entry site (IRES), which has a characteristic secondary 

structure and is required for viral translation. The HCV 3´-UTR is composed of a variable region, 

a poly(U/C) tract and the 3´X region and enhances the translation activity of the HCV IRES. The 

focus of this study was to analyze whether there is a communication between these two 

unique structures in the course of translation initiation and whether it depends on a cell type. 

To this end, we prepared chimeric constructs where the HCV IRES was replaced by the 

HCV-like IRES from the porcine teschovirus (PTV), a member of the Picornaviridae, or by the 5’ 

UTR of beta-actin mRNA. 

We could show after transfection of reporter RNAs into human hepatoma cells and into 

non-liver cells that the HCV 3´-UTR strongly enhances the reporter activity not only for 

translation directed by the HCV IRES but also by the PTV IRES and the 5’UTR of beta-actin 

mRNA, a typical cap-dependent messenger. Mutations in different regions of the HCV 3’-UTR 

decrease the stimulation effect. A similar enhancement of the IRES dependent expression 

could be seen by replacing the HCV 3’-UTR with a polyA-tail. The nature of the observed 

stimulation effect of the HCV 3’ UTR on the expression of mRNAs with quite different modes of 

translation initiation will be discussed.



ANDRZEJ NIERADKA 

Interaction between Grsf1 and elements within alternative exon of SCF 

sensitive gene – Use1 affects translation 

Andrzej Nieradka 1, Marleen Hameete 1, Marieke von Lindern 1, Christopher Ufer 2, Hartmut 

Kühn 2 

1 Erasmus Medical Center, Netherlands 

2 University Medicine Berlin-Charité, Germany 

Erythroblasts can be expanded in vitro in presence of Epo, SCF and glucocorticoids, while they 

mature in presence of Epo only. SCF enhances proliferation of erythroblasts through activation 

of PI3K/mTOR and releases of eIF4E. We previously identified transcripts subject to SCF 

dependent polysome recruitment, and we showed that polysome recruitment of these 

transcripts is dependant on PI3K activity and eIF4E expression. The goal is to understand the 

mechanism of selective translation of these transcripts. One of the identified transcripts is 

Use1, encoding an atypical SNARE protein involved in retrograde Golgi-ER transport. We 

extended the 5’UTR of mouse Use1 using RACE at 65°C, which revealed the presence of two 

transcripts, due to alternative splicing of an intron in the 5’UTR. The 5’UTR contains several 

uORF and a strong secondary structure upstream of the alternatively spliced intron (ASI), which 

deleted from reporter constructs has a major effect on in vitro translation of the transcript, but 

only a minor effect on translation in hematopoietic cells. Interestingly, the unspliced transcript 

was predominantly present in polysomes of erythroblasts. Accordingly, deletion of the ASI 

strongly impairs translation in vivo. Replacing the intron by unrelated sequences excluded the 

splicing process itself to be involved in effective Use1 translation. The intron sequence contains 

a G-rich sequence that appeared to bind G-rich sequence binding factor 1 (Grsf1). Grsf1 is a 

member of RNP family and plays a role on many levels of RNA processing. Increased 

expression of Grsf1 stimulated translation of the Use1 reporter, which was abrogated when the 

G-rich sequence was deleted from the reporter. Consistently, knock-down of Grsf1 in 

erythroblast cells decreased Use1 protein expression without affecting Use1 transcription. In 

conclusion, Grsf1 appears to control Use1 expression. The role of Grsf1 and Use1 in 

erythropoiesis is under investigation. 

204

DIERK NIESSING 

205 


An Extended RNA-binding Surface of She2p Oligomers is Required for mRNP 

Assembly and Localization 

Dierk Niessing 1, Marisa Müller 1, Ralf-Peter Jansen 2 

1 Helmholtz Zentrum München, Germany 

2 University of Tübingen, Germany 

In eukaryotic cells, dozens to hundreds of different mRNAs are localized by specialized 

motor-dependent transport complexes. We addressed the open question how the 

RNA-binding protein She2p of a yeast mRNA-transport complex specifically binds to several 

transcripts and how nuclear mRNA binding influences cytoplasmic transport-complex function. 

Previous results suggested that She2p is dimeric in solution. In this study, we determined that 

She2p forms tetrameric complexes that are required for mRNA binding in vitro and transcript 

localization in vivo. We further observed that She2p has an only moderately higher affinity for 

localizing transcripts when compared to unrelated stem-loop RNAs. However, for specific 

binding to localizing transcripts She2p requires an extended RNA-binding surface, whereas for 

unspecific RNA binding fewer surface features are sufficient. Mutation of the extended She2p 

surface selectively impairs in-vitro binding to localizing RNAs, cytoplasmic mRNP assembly in 

vivo, and its subsequent mRNA localization. The lack of a strong difference in affinity to 

mediate selectivity might be due to She2p’s requirement to recognize dozens of target RNAs in 

the nucleus with similar, yet distinct RNA-folding features. However in the cytoplasm, RNAs 

bound by She2p via its extended RNA-binding surface assemble with She3p into stable, highly 

specific transport complexes. In summary, our study shows that specificity at early, nuclear 

steps of complex assembly is not primarily mediated by higher affinity. In addition, we provide 

evidence for a close mechanistic dependence of cytoplasmic mRNP assembly on the spatially 

separated, preceding nuclear mRNA binding events by She2p.



EMILY NIKOLIC 

Programmed ribosomal frameshifting: a structural approach 

Emily Nikolic 1, Ian Brierley 1, John Flanagan 2, Robert Gilbert 2 



Many viral mRNAs contain programmed -1 ribosomal frameshifting signals that instruct 

ribosomes to change reading frame at a defined point. Two mRNA components are required: a 

heptanucleotide slippery sequence where frameshifting occurs, and a stimulatory RNA 

structure, typically a pseudoknot but occasionally a stem-loop. The mechanism of 

frameshifting is not fully understood, but likely involves direct modulation of the elongation 

cycle by stimulatory structures. The cryo-EM structure of ribosomes paused at the infectious 

bronchitis virus (IBV) pseudoknot revealed characteristic features: a bent P-site tRNA, the 

presence of eEF2 and mRNA entry-channel density likely attributable to the pseudoknot. To 

assist in assigning this density, we inserted a hairpin of known structure from phage PP7 into 

loop 3 of the IBV pseudoknot and purified ribosomes stalled at this structure for cryoEM. 

Reconstructions of these complexes reveal a modified entry-channel density, consistent with 

the view that the pseudoknot is present at this position. Additionally, we are characterizing 

ribosomes paused at diverse stimulatory structures of known three-dimensional architecture – 

the beet western yellows virus (BWYV) and simian retrovirus-1 (SRV-1) pseudoknots and the 

HIV-1 two-stem helix – using cryoEM and biochemical techniques. We demonstrate that all 

these structures pause a significant proportion of translating ribosomes, sufficient for isolation 

of frameshift-intermediates for structural analysis. Preliminary cryo-EM reconstructions are 

underway, and higher resolution analysis may further elucidate the mechanistic details of -1 

frameshifting. 

206

MARTIN OTT 

207 



mitochondrial translation 

Martin Ott, Johannes M. Herrmann, Martin Prestele 

University Kaiserslautern, Germany 

During evolution, many proteins of the mitochondrial ribosome acquired additional domains 

pointing at specific properties or functions of the translation machinery in mitochondria. Here, 

we analyzed the function of Mrpl36, a protein associated with the large subunit of the 

mitochondrial ribosome. This protein, homologous to the ribosomal protein L31 from bacteria, 

contains a mitochondria-specific C-terminal domain that is not required for protein synthesis 

per se, however, its absence decreases stability of Mrpl36. Cells lacking this C-terminal 

domain can still synthesize proteins, but these translation products fail to be properly 

assembled into respiratory chain complexes and are rapidly degraded. Surprisingly, 

overexpression of Mrpl36 appears to even increase the efficiency of mitochondrial translation. 

Our data suggest that Mrpl36 plays a critical role during translation that determines the rate of 

respiratory chain assembly. This important function appears to be carried out by a stabilizing 

activity of Mrpl36 on the interaction between large and small ribosomal subunits which could 

influence accuracy of protein synthesis.



XOCHITL PEREZ-MARTINEZ 

Pet309 is a multidomain protein involved in translational activation of the COX1 

mRNA in Saccharomyces cerevisiae mitochondria 

Xochitl Perez-Martinez, Faviola Tavares-Carreon, Angelica Zamudio-Ochoa, Yolanda 

Camacho-Villasana 

Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Mexico 

Pet309 belongs to the mitochondrial translational activators family. It is necessary for 

translation of the COX1 mRNA. Cox1 is the largest subunit of cytochrome c oxidase, the last 

electron acceptor of the respiratory chain. Cox1 is encoded by the mitochondrial COX1 gene, 

and is synthesized within matrix ribosomes. Pet309 acts on the COX1 mRNA 5'-UTR to 

activate translation, however very little is understood about the mechanisms of action of this 

protein. Genetic evidence suggests that mitochondrial translational activators bind to the 

5'-UTR of their target mRNA and to the ribosomal small subunit to tether translation initiation to 

the mitochondrial inner membrane. The sequence of Pet309 predicts at least 2 different 

domains. The central portion bears 8 to 12 PPR (pentatricopeptide repeat) domains, which are 

specific for mitochondria and chloroplast proteins. Most of the PPR proteins are involved in 

RNA metabolism, however their mechanism of action is not understood. Pet309 has a 200 

amino acid C-terminal region, which has no identified motifs, but is highly conserved among 

fungi. To study the mechanism of action of Pet309 we first created deletion mutants of either 

the PPR or the C-terminal regions of Pet309. Both portions are necessary to support COX1 

translation, however the C-terminal portion is in addition involved in stabilization of the COX1 

mRNA. Even though over expression of the mutant Pet309 proteins lead to over accumulation 

of the COX1 mRNA, the messenger was not translated. Both deletion mutants were able to 

bind to the COX1 mRNA 5'-UTR in vivo, as judged by Pet309 immunoprecipitation, RNA 

isolation and cDNA analysis. By similar experiments we observed interaction of the mutants 

with the mitochondrial 15s rRNA. Taken together our results suggest that both, the PPR 

portion and the C-terminal end of Pet309 are necessary for interaction with the ribosome and 

with the COX1 5'-UTR, but in addition the C-terminal end is involved in COX1 mRNA stability. 

208

NICOLA PHILLIPS 

Single Molecule Investigations of eukaryotic Initiation Factor 4A 


209 


NM Phillips, A Bottley, S Allen, KA Spriggs University of Nottingham, UK Eukaryotic Initiation 

Factor (eIF) 4A is the most abundant initiation factor and the prototypical member of the 

DEAD-box family of helicases. Once recruited to the cap-binding complex, eIF4F, eIF4A 

unwinds inhibitory RNA secondary structure in the 5’ untranslated region (UTR) of mRNAs, 

promoting efficient ribosomal scanning to the start codon. The requirement for eIF4A in 

translation initiation correlates with increasing 5’ UTR length, suggesting that regulating the 

activity of eIF4A may affect the translation of particular mRNAs. It is well established that the 

transcripts of genes involved in cell cycle control and proliferation have long 5’ UTRs; therefore 

altering the activity of eIF4A may affect these genes specifically. Single molecule studies of 

motor proteins enable detailed mechanistic analysis that bulk studies cannot achieve. This 

project will utilise single molecule techniques to further understand the mRNA sequence 

specific requirement of eIF4A and its accessory protein eIF4B. Optical tweezers utilise light in 

the form of a focussed laser beam to ‘trap’ and manipulate particles, such as nucleic acid 

chains tethered between two beads. By trapping an RNA:DNA construct containing a central 

stemloop hairpin known to be inhibitory to ribosomal scanning, we are able to observe 

changes in length to the molecule as eIF4A unwinds the secondary structure. Once 

established, this single molecule system will be used to observe eIF4A activity with its 

accessory protein eIF4B and known eIF4A inhibitors. Selected 5’ UTRs from mRNAs identified 

as requiring eIF4A from microarray screens will be used to investigate the behaviour of eIF4A 

on specific transcripts.



PHILIPPE PIERRE 

Investigating the role of translation regulation in dendritic cells 

CNRS, France 

Emerging evidences indicate that translation regulation plays a major role in immunity and its 

dysfunction can lead to important diseases. Moreover, we recently observed that protein 

synthesis is tightly regulated in Dendritic Cell (DC), one of the key coordinator of the immune 

system. We are currently interested in obtaining a global view of translational regulation in DC 

and in defining the functional importance of this regulation for immunity. We are using different 

approaches including to define the nature of mRNA molecules engaged or not in translation at 

various stages of DC activation by microbes. We have isolated polysomal-bound mRNA, 

microRNAs, and total mRNA populations and characterized them using DNA and RNA 

microarrays. A comparative and statistical analysis has allowed us to define genes, which are 

translationaly regulated during DC activation by pathogens and have not yet been implicated in 

immunity. Moreover, using the aminonucleoside antibiotic puromycin and newly generated 

anti-puromycin monoclonal antibodies, we have developed an alternative and sensitive solution 

to visualize and monitor translation in single and mixed populations of live cells (SUnSET). We 

provide evidence with this technology of the importance of translation regulation during the 

antigen specific activation of naïve T cells by DCs and the production of cytokine by DCs. 

Using this technology we can also purify specifically newly synthesized proteins and identify 

them through mass spectrometry. 

210

JULIE PILOTTE 

211 


Expression levels of the cold-stress inducible RNA-binding motif 3 protein, 

RBM3, alter the outcome of microRNA processing 

Julie Pilotte, Peter Vanderklish 

The Scripps Research Institute, United States of America 

A wide variety of cellular events, including proliferation, differentiation, and adaptive responses 

to stress, require de novo protein synthesis. MicroRNAs (miRNAs) – a diverse family of small, 

non-coding RNAs that mediate translational inhibition – play a major role in the specificity and 

timing of translational responses involved in such events. We have found that the RNA-binding 

motif protein 3 (RBM3), a member of the cold-inducible and glycine rich family of 

mRNA-binding proteins, has profound effects on the biogenesis of miRNAs. Previously, we 

reported that overexpression of RBM3 leads to an overall increase of translation in neuronal 

cell lines, and to a decrease in a miRNA-containing fraction resolved on sucrose gradients. 

Further analysis has revealed that altering RBM3 levels modifies post-transcriptional regulation 

of miRNA biogenesis and has widespread and bidirectional effects leading to significant 

changes in mature miRNA levels. Reciprocal effects on individual miRNAs are obtained by 

knockdown and overexpression of the protein. Northern blot analysis of precursor and mature 

transcripts indicates that changes in ~22mer species are likely due to altered processing of pri- 

and pre- transcripts. We also find that RBM3 associates with and regulates the levels of 

miRNA processing enzymes. Importantly, changes induced by experimental overexpression of 

RBM3 are reproduced by induction of RBM3 by cold shock. Although RBM3 was originally 

characterized as a cold-inducible protein, it is induced by many other physiological stressors, 

and we find that its expression is developmentally regulated, showing high levels in proliferating 

and differentiating neurons. The fact that RBM3 can be upregulated by several types of 

stresses and during development suggests that it could play a fundamental role in regulating 

the profile of expressed miRNAs to achieve tailored, adaptive translational responses.



HUGO PINHEIRO 

Allele Specific CDH1 Down-Regulation Increases Susceptibility to Diffuse 

Gastric Cancer 

David Huntsman 1, Hugo Pinheiro 2, Renata Carriço 2, Joana Carvalho 2, Patrícia Oliveira 2, 

Leonor Gusmão 2, Susana Seixas 2, Raquel Seruca 2, Carla Oliveira 2 

1 BCCA, Canada 

2 IPATIMUP, Portugal 

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant disease characterized by 

clustering of early-onset diffuse gastric cancer (DGC). In 40% of the cases, E-cadherin (CDH1) 

heterozygous germline alterations segregate with the disease. Independently of CDH1 

alterations, almost all DGC display mislocalized or absent E-cadherin immunoexpression. The 

cause of DGC in families without known alterations may be due to new germline epi/genetic 

defects at the CDH1 locus. We aimed at determining whether CDH1 negative HDGC patients 

display germline CDH1 allelic expression imbalance, using a Single Nucleotide Primer 

Extension based procedure and tried to uncover the genetic abnormality leading to CDH1 

hemizygous expression. CDH1 ASE analysis was performed in PBL’s RNA using two 

polymorphic sites from 21 cancer-free individuals and 27 HDGC probands (9 carrying CDH1 

mutations). Germline CDH1 promoter methylation, deletions and haplotype related 

susceptibility was analysed in these patients. ASE analysis showed equivalent expression of 

both CDH1 alleles in cancer-free individuals and monoallelic CDH1 expression or allelic 

imbalance (AI) in all CDH1 mutant HDGC probands and over 80% of HDGC CDH1 negative 

probands (p=3,56E-06). Germline deletions and promoter hypermethylation were found in 3 

probands displaying AI. We failed to find a risk haplotype associated with AI. CDH1 AI is highly 

frequent among CDH1 negative HDGC probands and allowed the identification of germline 

hypermethylation and deletions in 3 patients. Our results show that patients with CDH1 AI 

display high risk of developing HDGC. 

212

TERRA-DAWN PLANK 

213 


Identification of RNA binding proteins to the HIV-1 5’ leader: insights into 

mechanisms of translation initiation 

Terra-Dawn Plank, Christopher Abraham, Jeffrey Kieft 

University of Colorado at Denver Anschutz Medical Campus, United States of America 

The 5’ leader of the HIV-1 genomic RNA is highly structured with many domains that are 

critical to the viral life cycle. One function of this 5’ leader is the recruitment of the host cell 

translation machinery for synthesis of viral packaging proteins. It has been proposed that the 

HIV-1 5’ leader uses both cap-dependent and cap-independent translation initiation strategies; 

however, the mechanism of this dual initiation, as well as the contribution of each mechanism 

to the viral life cycle, has yet to be determined. To begin investigating these unknowns, we are 

employing a pull-down assay coupled with mass spectrometry for identification of cellular 

proteins which bind the 5’ leader. Using an uncapped 5’ leader, we have identified both 40S 

and 60S ribosomal proteins, as well as a subset of eukaryotic translation initiation factors, as 

potential binders of the 5’ leader. We also have identified a population of nuclear proteins 

which pull-down with the 5’ leader in a cell cycle-dependent manner. Interestingly, many of 

the proteins identified in our studies are also critical to viral replication; however, the 

mechanism by which they mediate their effects is unknown. Taken together, our preliminary 

data support a possible role for cap-independent translation during virally induced G2/M 

arrest. Our studies begin to cast light on the mechanisms of translation initiation from the 

HIV-1 genomic RNA and may also reveal RNA-protein interactions critical for steps in the viral 

life cycle other than translation.



MICHAEL POWELL 

Further characterisation of the translational termination-reinitiation signal of 

influenza B segment 7 

Richard Jackson, Michael Powell, Kendra E. Leigh, Tuija Poyry, T. David K. Brown, Ian 

Brierley 


Termination-dependent reinitiation is a translational mechanism used to co-ordinately regulate 

expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B 

segment 7 RNA. The AUG start codon of the BM2 ORF overlaps the stop codon of the M1 

ORF in the pentanucleotide UAAUG and about 10% of ribosomes terminating at the M1 stop 

codon go on to reinitiate translation at the overlapping AUG codon. BM2 synthesis is 

dependent on translation through a 45nt stretch of RNA immediately upstream of the UAAUG 

overlap termed the termination upstream ribosome binding site (TURBS). This region may act 

to tether 40S ribosomal subunits to the mRNA following termination and a short region of the 

TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this 

process. We provide further evidence to support a direct interaction beween mRNA and rRNA 

using antisense oligonucleotide targeting and functional analysis in yeast cells. We also show 

that the position of the TURBS with respect to the UAAUG overlap is crucial, and that 

termination too far downstream of the 18S complementary sequence inhibits this process, 

probably due to reduced 40S tethering. However, in reporter mRNAs where the start codon 

alone is moved downstream, termination-reinitiation efficiency is reduced only about two-fold, 

suggesting that the 40S subunit may regain the ability to scan after termination. Mutational 

analysis reveals that the entire TURBS is necessary for termination-reinitiation and is split into 

primary sequence-specific and sequence-independent elements. In addition to its likely role in 

tethering 40S subunits post-termination, the TURBS also binds eIF3. Here we show that this 

protein stimulates reinitiation from both wild-type and defective TURBS when added 

exogenously to translations. How eIF3 acts to stimulate reinitiation is not known, but potential 

mechanisms are discussed. 

214

TUIJA POYRY 

215 


The mechanism of internal ribosome entry and initiation site selection on the 

FMDV IRES 

Richard Jackson, Tuija Poyry 


Foot-and-mouth disease virus (FMDV) has a structurally very similar IRES to cardioviruses, but 

the FMDV IRES has two functional AUG codons, 84 nt apart, producing two forms of 

L-protein, Lab and Lb. The relative initiation frequency at the two sites differs between FMDV 

strains ranging from 30 to 50% at the Lab-site and from 50 to 70% at the Lb-site. This 

difference can be partly explained by the different nucleotide sequence context around the two 

sites, which is generally poor around the Lab-site and generally very good around the Lb-site. 

However, if the bulk of the IRES is deleted resulting in a construct that has a 86nt long 5’UTR 

which can be used for cap-dependent translation, the Lab site is used about twice as 

frequently as the Lb site. As antisense oligonucleotides spanning the Lab site inhibit initiation at 

both AUGs, while an antisense oligonucleotide covering the Lb site induces initiation at a 

non-AUG codon between the two AUGs, it is thought that all ribosomes enter at or near the 

Lab site, but only a minority initiate there, while the rest scan to the Lb site. Mutation of the Lab 

AUG decreases translation about 40 %, but it has no negative effect on initiation at the Lb site 

and may even be stimulatory. Replacing the sequence directly downstream of the Lab site with 

a reporter decreases translation about 50 %, but keeping 12 nt of the FMDV coding sequence 

recovers to 75 % and 48 nt restores translation to the wt level. Similarly, having a stem-loop 

immediately after the Lab site abolishes almost all translation, but the IRES activity can be 

recovered to about 70% by keeping 12 nt of the coding sequence, although the Lab site is 

only used at 10 % efficiency relative to the wt IRES. In addition, deleting 21 nt after the Lab site 

also decreases translation, but does not affect the start site selection. Current experiments try 

to establish what is the functional role of the coding region required for the fully active FMDV 

IRES.



MARTIN PRESTELE 


mitochondrial translation 

Martin Prestele 1, Martin Ott 1, Johannes M. Herrmann 2 

1 TU Kaiserslautern, Germany 

2 University Kaiserslautern, Germany 

The complexes of the respiratory chain represent mosaics of nuclear and mitochondrially 

encoded components. The processes by which synthesis and assembly of the various 

subunits are coordinated remain largely elusive. During evolution, many proteins of the 

mitochondrial ribosome acquired additional domains pointing at specific properties or functions 

of the translation machinery in mitochondria. Here, we analyzed the function of Mrpl36, a 

protein associated with the large subunit of the mitochondrial ribosome. This protein, 

homologous to the ribosomal protein L31 from bacteria, contains a mitochondria-specific 

C-terminal domain that is not required for protein synthesis per se, however, its absence 

decreases stability of Mrpl36. Cells lacking this C-terminal domain can still synthesize proteins, 

but these translation products fail to be properly assembled into respiratory chain complexes 

and are rapidly degraded. Surprisingly, overexpression of Mrpl36 appears to even increase the 

efficiency of mitochondrial translation. Our data suggest that Mrpl36 plays a critical role during 

translation that determines the rate of respiratory chain assembly. This important function 

appears to be carried out by a stabilizing activity of Mrpl36 on the interaction between large 

and small ribosomal subunits which could influence accuracy of protein synthesis. 

216

MAYA PRIZANT 

Cytoskeletal Control of c-Jun Translation 

Maya Prizant, Iris Ben-Dror, Andrea Atzmon, Keren Merenbakh, Lily Vardimon 

Tel Aviv University, Israel 

217 


The cytoskeleton is a dynamic network that undergoes restructuring during cellular events, 

influencing cell proliferation, differentiation and apoptosis. We have shown that accumulation of 

c-Jun, a member of the AP1 family of transcription factors that play a key role in normal and 

aberrant cell growth, dramatically increases upon depolymerization of the cytoskeleton, and 

that, unexpectedly, this increase is controlled translationally. The cytoskeletal-dependent 

increase in c-Jun translation is mediated by the untranslated regions (UTRs) of the c-Jun 

transcript, in particular by the 5'UTR, which is exceptionally long, conserved during evolution 

and have the potential of forming stable secondary structures. The cytoskeletal-dependent 

increase in c-Jun translation is not due to alternative splicing or a change in the cellular 

localization of the c-Jun transcript. Deletion analysis showed that the first 277 bases of the 

c-Jun 5'UTR are sufficient to mediate the cytoskeletal dependent control of c-Jun translation. 

RNA pull down assays revealed the presence of a cytoplasmic protein that binds this region 

specifically. The bound protein was identified by mass spec analysis as glycyl-tRNA synthetase 

(GlyRS). RNA secondary structure analysis using Mfold software predicted that the 277 bases 

of the c-Jun 5'UTR can form a stable structure with three hairpin motifs. Deletion analysis 

demonstrated that only one of the hairpin motifs binds GlyRS and mutation analysis identified 

three nucleotides in the loop that are essential for binding. Co-Immunoprecipitation analysis 

revealed the presence of two cytoplasmic protein bands that bind GlyRS specifically. Our 

findings suggest that restructuring of the cytoskeleton activates a signaling pathway that 

affects RNA binding protein(s) and alters thereby the translatability of the c-Jun transcript. This 

novel mechanism of c-Jun regulation might be relevant to physiological condition in which 

c-Jun plays a pivotal role.



ALESSANDRO QUATTRONE 

Widespread uncoupling between transcriptional and translational control of 

gene expression in mammalian cells 

Enrico Blanzieri 1, Alessandro Quattrone 2, Toma Tebaldi 2, Gabriella Viero 2, Alessandro 

Provenzani 2, Angela Re 2 

1 Department of Information Engineering and Computer Science, University of Trento, Italy 

2 Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento, 

Italy 

Transcriptome analysis by total mRNA profiling provides a measurement of the degree of 

variation for each single mRNA species after a cell state transition. It is becoming a general 

notion that variations in protein levels do not necessarily correlate with variations in total mRNA 

levels, for the presence of post-transcriptional controls which influence translational fitness of 

mRNAs. Nevertheless, the extent of this phenomenon and the rules, if any, governing it are still 

completely unknown. 

To address this issue we took advantage of a number of studies performed using polysomal 

mRNA profiling in combination with classical total mRNA profiling in different mammalian 

systems. A normalization of the raw data coming from these datasets and a statistical 

meta-analysis aimed at maximizing uniformity in data processing have been performed. From 

the comparison of the results a general, profound uncoupling between transcriptional and 

translational variations of mRNA levels emerges. It seems clear that virtually each change in 

cytoplasmic mRNA steady-state level is subjected to a further elaboration by a 

post-transcriptional decision program, leading in several cases to a widespread buffering of the 

cytoplasmic changes which transfers only a small fraction of them to translation. 

A model characterized by a mRNA storage compartment is proposed to explain how a 

change in translational fitness can counteract or magnify a parallel change in citoplasmic 

mRNA availability. 

218

ANABELA RAMALHO 

A cystice fibrosis mutation that unravels alternative translation initiation: 

structural, functional and clinical implications 

219 


Carlos Farinha 1, Filipa Mendes 1, Margarida D Amaral 1, Celeste Barreto 2, Juan Gonçalves 3, 

Anabela Ramalho 4 

1 Faculty of Sciences, University of Lisbon, Portugal 

2 Hospital de Santa Maria, Portugal 

3 Hospital do Divino Espírito Santo, Portugal 

4 National Institute of Health Dr. Ricardo Jorge, Portugal 

Mutations in the CFTR gene cause Cystic Fibrosis (CF) the most common life-threatening 

autosomal recessive disease affecting Caucasians. Here, we report a novel CFTR mutation 

(c.120del23) abolishing the normal translation initiation codon, which occurs in two Portuguese 

CF patients with classical CF. We investigated whether this deletion allows CFTR protein 

production (through usage of alternative internal codons) and whether the putative truncated 

CFTR form(s) are functional. 

Our data show that two shorter forms of CFTR protein are produced, indicating usage of 

internal initiation codons. Mutants of four methionine codons downstream to M1 (M82, M150, 

M152, M156) were generated to determine which is mostly used to initiate CFTR translation. 

Analysis of the proteins resulting from individual and combined mutants, revealed that each of 

the M150/M152/M156 codons can mediate alternative translation of the c.120del23-CFTR 

construct. Moreover, functional characterization of the resulting N-truncated protein(s) showed 

that at least one of these proteins reaches the cell membrane, however exhibiting drastically 

reduced Cl- channel activity. Furthermore, pulse-chase studies show that the stability and 

processing efficiency of these CFTR N-truncated proteins are significantly reduced vs 

wt-CFTR, indicating an important role for the N-terminus in avoiding CFTR turnover and 

ensuring correct plasma membrane trafficking. 

Overall, our results indicate that the M150/M152/M156 codons can generate truncated CFTR 

forms with decreased stability and processing efficiency, together with drastically reduced 

function. These data correlate well with the severe clinical CF phenotype of these patients.



JAANUS REMME 

Ribosome reactivation by replacement of damaged proteins 

Jaanus Remme, Arto Pulk, Aivar Liiv, Ülo Maiväli, Lauri Peil 

University of Tartu, Estonia 

Ribosomal functions are vital for all organisms. rRNA is susceptible to cellular RNases and 

ribosomal proteins are readily damaged by chemical modification under chemical stress. 

Accumulating damage to ribosomal RNA or proteins can disturb ribosome functioning. Inactive 

or partially active ribosomes could be degraded or possibly also repaired. Reactivation of 

chemically damaged ribosomes by protein replacement mechanism was studied in vitro and in 

vivo. Ribosomes were inactivated by modification of Cys residues. Incubation of modified 

ribosomes with total ribosomal proteins led to reactivation of translational activity. Ribosomal 

proteins split by LiCl are equally active in restoration of ribosome function. Incubation of 70S 

ribosomes with radioactive r-proteins followed by separation of ribosomes identified 

exchangeable proteins. About 25% of total ribosomal proteins were found to be exchangeable. 

MS experiments using 15N r-proteins and non-labeled ribosomes revealed the different rate 

and extent of exchange of individual proteins. When stationary growth phase bacteria were 

exposed to radioactive amino acids specific ribosomal proteins were labeled. These shows 

that these r-proteins were incorporated into preexisting ribosomes and are therefore 

exchangeable in vivo. Similar sets of proteins were found to be exchangeable in vitro and in 

vivo under stress conditions in the stationary phase. We propose that repair of damaged 

ribosomes might be an important mechanism for maintaining protein synthesis activity under 

chemical damage. 

220

LUÍS RIBEIRO 

A new function for the Contactin associated protein 1, Caspr 1, in the 

regulation of GluR1 mRNA stability 

Sandra Santos, Ana Luisa Carvalho, Luís Ribeiro 

Center for Neuroscience and Cell Biology, University of Coimbra, Portugal 

221 


Glutamate receptors of the AMPA-type mediate fast excitatory synaptic transmission in the 

CNS and play key roles in synaptic plasticity. Little information exists about regulation of the 

transcripts for AMPA receptor subunits, which exist in dendrites, in close proximity to synaptic 

sites and strategically positioned for on-site protein synthesis. Our data show that Contactin 

associated protein 1 (Caspr 1), a cell adhesion molecule, increases GluR1 total protein levels 

when both proteins are co-expressed in COS 7 cells. Furthermore, Caspr 1 overexpression in 

neurons increased the endogenous levels of GluR1. The half-life of the GluR1 protein in COS 7 

cells was not affected by the presence of Caspr 1, excluding an effect of Caspr 1 on GluR1 

protein stability. Conversely, real-time qPCR studies showed that Caspr 1 co-expression 

increases the GluR1 mRNA levels, in a transcription-independent manner, and that this effect 

is mediated by the intracellular C-terminal domain of Caspr 1. As far as we know, this is the 

first evidence suggesting that the genetic expression of AMPA receptors can be regulated at 

the level of mRNA stability. Moreover, we demonstrated that simple deletion of 3’ UTR from 

GluR1 mRNA increases the levels of GluR1 mRNA, and that Caspr 1 has no effect on GluR1 

transcripts lacking the 3’UTR, suggesting that this region contains regulatory elements 

necessary for the effect of Caspr 1 on GluR1 mRNA levels. Indeed, multiple conserved 

canonical cis elements were found in the 3’UTR of GluR1 among which several binding 

sequences for RNA-binding proteins of the ELAV family. Taken together, our data indicate that 

Caspr1 plays a role in stabilizing GluR1 mRNA.



EMILIANO P. RICCI 

Untreated rabbit-reticulocyte lysate as an in vitro system to recapitulate 

translation inhibition driven by endogenous miRNAs as well as pre-miRNA 

processing 

Rachel Allison 1, Tuija Pöyry 1, Emiliano P. Ricci 2, Ricardo Soto Rifo 2, Taran Limousin 2, 

Didier Décimo 2, Theo Ohlmann 2, Richard Jackson 3 

1 Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK., United 

Kingdom 

2 Inserm U758 Ecole Normale Supérieure de Lyon, France 


Micro RNAs s (miRNAs) are small non-coding RNAs ( 22 bases long) encoded by the cell. 

Associated with the RNAi-induced silencing complex (RISC), miRNAs regulate gene expression 

by interacting generally with the 3’UTR of mRNAs and affecting their translation and/or stability. 

Here, we have developed an in vitro system to study the effects of endogenous miRNAs 

contained in the untreated rabbit reticulocyte lysate. For this, microarray profiling in the 

rabbit-reticulocyte lysate has been carried out and revealed that more than 550 miRNAs are 

present in this extract. Furthermore, the presence of RISC components such as Dicer, TRBP, 

Tudor-SN and Argonaute 2 was confirmed by western-blotting. Determination of the 

concentration of these miRNAs by quantitative RT-PCR revealed the prevalence of miR-451 

(which is expressed at high level during erythroid differentiation), together with miRNAs 

belonging to the let-7 family which are known to be present in most cell types. In order to test 

their effects on translation, several construct coding for the renilla luciferase were made bearing 

target sites for miR451 and let-7a. After preincubation of the corresponding RNAs with 

untreated rabbit reticulocyte lysate we could measure a 2-fold inhibition of luciferase 

expression compared to that of the control mRNA (bearing no target sites). Such an effect was 

not observed in the wheat-germ translation extract. This inhibition seems to occur at the 

translational level since no significant effect on mRNA stability could be detected by 

quantitative RT-PCR, and is not linked to a specific deadenylation of targeted mRNAs. 

Interestingly, mRNAs bearing perfect match target sites for miR-451 are not inhibited at the 

translational level but rather degraded indicating that the RISC machinery contained within the 

lysat is also able to recapitulate an siRNA response. 

Finally, we also show that the endogenous RISC is able to process miRNA precursors to fully 

mature miRNAs. Therefore, our finding validates the use of the untreated rabbit-reticulocyte 

lysate as a model in vitro system to study the mechanistics of translation regulation by miRNAs 

but also their processing. 

Work supported by a grant from the ANR 

222

ADELE RICCIARDI 

Identification of SLIP1 Binding Proteins 

Adele Ricciardi, Rachel Lerner, William Marzluff 

University of North Carolina- Chapel Hill, United States of America 

223 


The mRNAs encoding histone proteins in metazoans are the only eukaryotic mRNAs that do 

not end in a poly(A) tail, ending instead in a conserved stem-loop. This 3’ stem-loop is bound 

by stem-loop binding protein (SLBP), which is critical for all stages of the histone mRNA life 

cycle. Using a yeast two-hybrid screen with SLBP as bait, we identified a novel protein, 

SLBP-interacting protein 1 (SLIP1). SLIP1 interacts with the region of SLBP required for 

translation and with translation factor eIF4G. We further demonstrated that SLIP1 stimulates 

the translation of reporter mRNAs ending in a histone stem-loop and is required for optimal 

levels of histone protein synthesis in vivo. 

While it is clear that SLIP1 participates in histone mRNA translation, the entire cellular role of 

SLIP1 remains unknown. SLBP is critical for histone mRNA processing, translation and 

degradation, yet cells with knockdown of SLBP protein by RNAi remain viable, progressing 

slowly through the cell cycle and arresting in S-phase. Interestingly, knockdown of SLIP1 

causes rapid cell death, suggesting that SLIP1 plays a significant function necessary for 

viability aside from its role in histone mRNA translation. A yeast two-hybrid screen using SLIP1 

as bait was performed to identify SLIP1-interacting proteins and gain further knowledge about 

the function(s) of SLIP1. This screen identified proteins functioning in RNA metabolism, mRNA 

export, and a translation factor. To determine the biological significance of these protein 

interactions with SLIP1, we will use GST-pulldowns and directed yeast-two-hybrid to test 

which interactions are conserved across species. Additionally, we identified c-myc as a 

SLIP1-interacting protein and validated this interaction using GST pulldowns. Further studies to 

determine whether c-myc and SLIP1 interact in vivo and the function of this interaction will be 

presented.



VALENTINA RUGGERI 

eIF6 haploinsufficiency suppresses Myc oncogenic activity 

Valentina Ruggeri 1, Annarita Miluzio 1, Stefano Grosso 1, Stefano Biffo 1, 

Pier Carlo Marchisio 2 

1 San Raffaele Scientific Institute, Italy 

2 Vita Salute San Raffaele, Italy 

Eukaryotic initiation factor 6 (eIF6) affects cell cycle control and tumorigenesis. It was 

demonstrated that eIF6 is rate limiting for cell trasformation in vitro. In addition, sub-cutaneous 

injection of eIF6+/- mouse embryo fibroblasts MEFs transformed with DNp53+H-rasv12 

showed a reduction in tumor development in nude mice compared to wt mice. To study the 

role in tumorigenesis of eIF6 in vivo, we crossed eIF6+/- and wt mice with EμMyc transgenic 

(tg) mice, known as model of lymphoma in vivo. The EμMyc tg mice develop B cell lymphoma 

and manifest a mean survival time of approximately 6 months. The onset of lymphoma of 

eIF6+/- tg mice is markedly delayed compared to the wild type. In fact a significant percentage 

of eIF6+/- tg mice do not develop lymphomas even after one year of age. Moreover we find 

that eIF6+/- EμMyc tg mice exhibit a reduction in splenomegaly, the earliest syntom of the 

disease, and in tumor mass weight compared to the eIF6+/+ tg ones. Immunohistochemical 

and western blot analysis also demonstrate a reduction of both proliferating cells and 

hemopoiesis in the spleens of eIF6+/- EμMyc 4-5 weeks old mice. Preliminary data show a 

delay in G1/S phase progression of B cells of eIF6+/- tg mice, while the apoptotic rate is 

identical. Taken together, these data show that eIF6 haploinsufficiecy impairs Myc-induced 

lymphomagenesis. 

224

DAVIDE RUGGERO 

225 


Genetic dissection of oncogenic PI3K-Akt-mTOR pathway reveals 

deregulations in translational control via 4EBP1-eIF4E as a key determinant of 

cancer 

Oded Meyuhas 1, Davide Ruggero 2, Maria Costa 2, Andrew Hsieh 2, Ornella Zollo 2, Cole 

Davis 2, Kevan Shokat 2, Feldman Morris 2 

1 The Hebrew University-Hadassah Medical School, Israel 

2 University of California, San Francisco, United States of America 

The PI3K-Akt-mTOR signaling pathway is deregulated in the majority of human cancers. The 

mTOR kinase is activated by insulin, nutrients and growth factors and links these stimuli to 

control of ribosome biogenesis, protein synthesis, and cell growth downstream of PI3K-Akt 

signaling. Whether the ability of mTOR to modulate protein synthesis plays a causal role in 

tumorigenesis downstream of PI3K hyperactivation remains unknown. The eIF4E-binding 

proteins (4EBPs) and the S6 ribosomal protein (rpS6), are the most commonly employed 

readouts of PI3K-Akt-mTOR activation, yet their functional contribution to oncogenic PI3K 

signaling is unknown. We demonstrate that when protein synthesis rates are restored to 

normal levels in cancer-prone Lck-Akt transgenic mice, the majority of animals remain 

cancer-free suggesting that deregulations in translation control may be both necessary and 

sufficient to cause cancer. While phosphorylation of rpS6 is dispensable for cancer formation, 

eIF4E plays a critical function in control of protein synthesis rates, cell growth and cancer 

initiation downstream of PI3K activation. We show that eIF4E specifically controls cell survival 

but not proliferation, at least in part, through translational control of key anti-apoptotic proteins 

such as MCL-1. These findings suggest that cancer cells can hijack the nutrient and growth 

factor sensing function of PI3K-Akt-mTOR signaling used to modulate protein synthesis to 

promote cell growth and survival as a primary means for cellular transformation. While 

traditional mTOR inhibitors such rapamycin and associated rapalogues have limited clinical 

efficacy, here we show in-vivo that a novel ATP-active site inhibitor of mTOR that directly 

inhibits increases in eIF4E-dependent protein synthesis shows clinical promise. These findings 

provide a strong mechanistic rationale for pharmacological interventions that target specific 

components of the deregulated translational apparatus in cancer.



ARZU SANDIKCI 

Coordination of N-terminal enzymatic processing with co-translational folding 

and targeting of nascent polypeptides 

Arzu Sandikci, Günter Kramer, Matthias P. Mayer, Bernd Bukau 

ZMBH, Germany 

N-terminal enzymatic processing of nascent polypeptides is essential and conserved 

throughout evolution. While protein synthesis in the eukaryotic cytosol initiates with 

methionine, bacteria and eukaryotic organelles initiate translation with an N-formylated 

methionine. However, nearly all of the fully synthesized proteins lack the N-terminal formyl 

group. This is due to the action of the ribosome-associated peptide deformylase (PDF), which 

co-translationally removes the formyl group as the nascent chain emerges 

from the ribosomal exit tunnel. In the second step, the methionine aminopeptidase (MAP) 

catalyzes the removal of the N-terminal methionine. 

It has been shown that PDF binds to the 50S subunit of the E. coli ribosome through its 

C-terminal helical extension. This interaction localizes PDF close to the peptide exit tunnel for 

efficient co-translational removal of formyl residue from the N-terminal methionine of nascent 

polypeptides. Concurrent with these processes, decisions have to be made whether the 

nascent chain folds and resides in the cytoplasm or whether it is co-or post-translationally 

targeted to the membrane for translocation. These events take place very early in translation by 

the interactions of two ribosome-associated factors with the nascent polypeptide: the 

chaperone Trigger factor (TF) and the targeting factor signal recognition particle (SRP). 

Since all these processes need to occur in very early phases of translation while the nascent 

chain is just emerging from the exit tunnel, they must be temporally and spatially coordinated. 

We are investigating the molecular details of the dynamic interplay between the enzymes 

involved in N-terminal processing, PDF and MAP, the chaperone TF and the targeting factor 

SRP. 

226

BRUNO SARGUEIL 

Molecular mechanisms for HIV genomic RNA translation initiation 

Bruno Sargueil, Nicolas Locker, Nathalie Ulryck, Laurie James 

CNRS, France 

227 


Translation of the HIV-2 genomic RNA results in the synthesis of Gag, a large polyprotein that 

is cleaved to yield structural proteins. We have previously shown that three Gag isoforms are 

produced by an internal ribosomal entry site (IRES)-driven mechanism. The HIV-2 IRES 

element is entirely located within the Gag coding region and downstream the first AUG. 

Moreover, it directs the translation of the three Gag isoforms with no need for upstream 5' 

untranslated RNA sequence and this IRES element can recruit up to three independent 

initiation complexes onto one RNA molecule. 

To try and understand the molecular mechanisms involved in internal entry of the ribosome 

onto HIV genomic RNA we investigated the functional requirement for eIFs components, using 

affinity purification of 48S initiation complexes, in vitro reconstitution and known inhibitors of 

translation. 

Using affinity purified 48S complexes assembled onto the HIV-2 gag IRES RNA, we identified 

the initiation factors required during the initiation pathway, revealing the presence of eIF2, eIF3, 

eIF4A, eIF4G an eIF5, but devoid of eIF1 and eIF4E. We further demonstrate the formation of a 

binary complex between HIV-2 gag IRES and the 40S subunit, delineate the RNA motif 

responsible for this interaction, and also evidenced a direct interaction with eIF3, mediated by 

subunit eIF3b,c,d and f. Those properties are conserved among primate lentivirus. 

In parallel we have modelled the IRES secondary structure for HIV2, HIV1 and SIVmac, and 

shown by directed mutagenesis that this structure is responsible for the activity. 

At this stage the functional requirement for eIFs is reminiscent from those for picornavirus 

translation, while the isolated binding of eIF3 and 40S onto the HIV-2 IRES reminds the 

HCV-like pathway. 

Further work will focus on the 48S assembly pathway trying to unravel the role for the RNA 

structure, and for each protein factors.



EVELYN SATTLEGGER 

Gcn1 and actin binding to the central region of Yih1: Implications on Gcn2 

activation 

João A. R. G. Barbosa 1, Maria Carolina S. Moraes 2, Rafael M. Martins 2, 

Beatriz A. Castilho 2, Evelyn Sattlegger 3, Alan Hinnebusch 4 

1 Center for Structural Molecular Biology (CeBiME), Brazilian Synchrotron Light Laboratory, 

C.P. 6192, Campinas, Brazil 

2 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São 

Paulo, Brazil 

3 Institute of Natural Sciences, Massey University, Albany, New Zealand 

4 National Institutes of Health, United States of America 

In all eukaryotes, phosphorylation of eIF2a is a major mechanism for adjusting protein synthesis 

in response to a variety of environmental or intracellular stresses. The eIF2a kinase Gcn2 is 

crucial for overcoming amino acid starvation. For sensing starvation, the Gcn2 RWD domain 

must directly bind to its effector protein Gcn1. The mammalian protein IMPACT, highly 

expressed in neurons, and its yeast ortholog Yih1, consist of an Ancient Domain and an RWD 

domain with Gcn1 binding activity. We have shown that Yih1/IMPACT competes with Gcn2 for 

Gcn1 binding thereby diminishing Gcn2 activity. Deletion of YIH1 does not lead to constitutively 

active Gcn2, suggesting that Yih1 inhibits Gcn2 only under certain physiological conditions or 

in specific cellular compartments. We aim to further elucidate the cellular roles of Yih1/IMPACT. 

Our previous studies suggested that Yih1 resides in the cell in an inactive Yih1-G-actin 

complex, and when released from actin inhibits Gcn2. Supporting this idea, we show that 

Yih1-actin interaction is independent of Gcn1, and that Yih1-Gcn1 interaction does not require 

actin. Using various Yih1 fragments fused to GST, we found that the Yih1 RWD domain does 

not bind actin. Instead, actin binds the Yih1 central region. Surprisingly, the same Yih1 region 

binds Gcn1. The efficiency of the various Yih1 fragments on Gcn1/actin sequestration, and 

their effect on Gcn2 activity, will be discussed. In structural exercises we identified amino acids 

in Yih1 that may be involved in Gcn1 binding. Furthermore, we uncovered a conserved putative 

interaction surface characteristic for Ancient Domains that harbors determinants characteristic 

to either eukaryotes or prokaryotes. These results reinforce our model for a universal role of 

Yih1/IMPACT in a cross-talk between the cytoskeleton and translation, and likely relevant in 

neurons given that mammalian Gcn2 is also involved in memory formation and behavior. 

228

CLAUDIA SCHECKEL 

229 


The STAR-domain protein GLD-1 stabilizes mRNAs in the C. elegans germ line 

Friedrich Miescher Institute, Switzerland 

The GLD-1 protein, which is related to mammalian Quaking and Sam68, is critical for many 

developmental decisions during worm germline development. GLD-1 has been shown to 

repress translation of mRNAs encoding various proteins, such as Notch/GLP-1 or CEP-1/p53. 

To address the mechanism of GLD-1 mediated repression, we have examined the ribosomal 

association of several GLD-1 target mRNAs using polysome profiles. We found that GLD-1 

targets are mostly present in pre-monosomal fractions, suggesting that translation initiation of 

these mRNAs is inhibited. Surprisingly, the knock down of GLD-1 does not lead to a 

re-distribution of its target mRNAs in a polysome profile, but results in their reduced 

abundance. This suggests that only a minor fraction of each target mRNA is abnormally 

translated in the GLD-1 (-) germ line, and that GLD-1 stabilizes associated mRNAs. We find 

that this stabilization of at least one GLD-1 target also requires the conserved RNA helicase 

CGH-1/Dhh1, which has been previously suggested to stabilize various maternal mRNA but 

not GLD-1 targets. At this time, the precise functional relation between GLD-1 and CGH-1 

remains unclear. However, the closest mammalian GLD-1 ortholog, Quaking, has been 

implicated in stabilizing at least one mRNA. Thus, a primordial role of GLD-1/Quaking-like 

proteins may be to protect associated mRNA from degradation, perhaps through packaging 

into RNP granules.



LUCA SCHENK 

Global Identification of Potential RNA Targets for the Paralogous La-related 

RNA binding Proteins Slf1p and Sro9p 

Luca Schenk, André Gerber 

ETH, Switzerland 

Despite the importance of RNA binding proteins (RBPs) in translational control of specific 

mRNAs, to date, the RNA targets of the numerous known or predicted eukaryotic RBPs are 

largely unknown. By means of DNA microarrays we aim at determining the RNA targets of the 

two evolutionary conserved La-related yeast proteins Slf1p and Sro9p. Both have been shown 

to bind RNA in vitro and to preferentially associate with polysomes, which makes them prime 

candidates to be mRNA translation-regulating RBPs. 

Our experiments show that Slf1p and Sro9p bind to a largely overlapping set of hundreds of 

mRNAs. In contrast to the bona fide La protein, which binds to the 3’polyuridine tail of nascent 

polymerase III transcripts, we did not find significant association of noncoding RNAs. Among 

the common RNA targets are most transcripts coding for components of the cytoplasmic 

ribosome, the histones as well as many mRNAs of genes involved in secretion. Interestingly, 

we also find transcripts that are involved in copper homeostasis which may link to previous 

findings that implicate Slf1 in copper detoxification. Indeed, we could confirm that 

overexpression of SLF1 leads to enhanced growth in elevated copper concentrations. 

Moreover, we were able to show that this phenotype is likely to depend on the RNA binding 

capacity of protein since mutations in La-domain residues known to be crucial for RNA binding 

of the La protein, abolished the copper phenotype. Finally, microarray and Real-Time-PCR 

analysis of RNA isolated from SLF1 overexpressing and deleted cells suggests that Slf1 

specifically stabilizes its RNA targets. We currently extend this analysis to SRO9 mutant and 

overxpressing strains with a special focus on the small fraction of targets that are distinct 

between Slf1p and Sro9p, since they might explain the observed SLF1 copper phenotype, 

which is not manifested in SRO9 overexpressing cells. 

230

GERT SCHEPER 

Mutations in the genes encoding the eIF2B subunits lead to abnormal 

maturation and function of glial cells in the white matter of the brain 

231 


Elly Hol 1, Steve Goldman 2, Gert Scheper 3, Ilja Boor 3, Marianna Bugiani 3, Nienke Postma 3, 

Barbara van Kollenburg 3, Marjo van der Knaap 3 

1 3Netherlands Institute for Brain Research, Amsterdam, Netherlands 

2 University of Rochester, Rochester, NY, United States of America 

3 VU University Medical Center, Amsterdam, Netherlands 

Vanishing white matter disease (VWM) is one one the most prevalent childhood 

leukoencephalopathies. It is caused by mutations in any of the genes encoding the five 

subunits of the eukaryotic translation initiation factor eIF2B. Like other white matter diseases, 

VWM is characterized by progressive cerebellar ataxia and less prominent spastisticy. 

However, typical to VWM are episodes of major neurological deterioration triggered by minor 

stresses as fever and mild trauma. Neuropathological features of VWM are also distinctive. The 

episodes of major deterioration correlate pathologically with the appearance and extension of 

cystic rarefaction and cavitation of the white matter, in which dysmorphic astrocytes and 

insufficient formiation of scar tissue (gliosis) are a major feature. Around cavitated areas, 

hypomyelination and myelin loss coexist with a striking increase in the density of 

oligodendrocytes. These findings suggest that a functional impairment and/or a maturation 

defect of macroglial cells might contribute to the pathogenesis of VWM. 

We demonstrate delayed maturation of astrocytes and oligodendrocytes in the brain of 5 

VWM patients using immunohistochemistry, Western blot, microarray analysis, and qPCR. We 

show that astrocytes do proliferate in VWM, but do not reach full maturity and overexpress the 

delta isoform of the cytoskeletal protein GFAP together with the stress protein alpha 

B-crystallin, a finding that might explain their abnormal morphology and dysfunction. 

Oligodendrocytes are immature in VWM, and many fail to go beyond the status of progenitors. 

However, mRNAs of major myelin proteins are normally detected, although to a lesser degree 

than in normal controls, suggesting that the myelinogenic program per se is not perturbed in 

VWM, but expression of myelin proteins is impaired. This combination of findings is not found 

in controls and other genetic white matter disorders, and thus appears to be specific for VWM.



T. MARTIN SCHMEING 

Structural Studies of Translation Initiation 

Jon Lorsch 1, T. Martin Schmeing 2, V Ramakrishnan 2, Lori Passmore 2 

1 Johns Hopkins University School of Medicine, United States of America 

2 LMB, Cambridge, United Kingdom 

Initiation of translation is the process by which an initiator tRNA and the start codon of mRNA 

are positioned into the ribosomal P site to allow protein synthesis. In eukaryotes, initiation 

requires at least 12 eukaryotic initiation factors (eIFs), composed of 28 different polypeptides. 

Many molecular mechanisms of translation initiation remain unclear. One of the first steps 

involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. 

Using cryo-electron microscopy (cryo-EM), we have obtained 3D reconstructions of the 40S 

subunit bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that 

together, eIF1 and eIF1A stabilise a conformational change that opens the mRNA binding 

channel, making the 40S competent for binding of mRNA and tRNA, and for scanning. We 

have obtained cryo-EM images of the mRNA, the tRNA ternary complex (eIF2 – GTP – 

met-tRNA) and eIF5 bound to the 40S-eIF1-eIF1A initiation complex. There is significant 

conformational and compositional heterogeneity in the sample, which we are addressing using 

biochemical and computational means. Our maps reveal the binding site of the ternary 

complex, and the extent of conformational changes in the 40S ribosome required for mRNA 

binding. 

232

EDUARD SCHREINER 

Mechanistic insights into quality control by the ribosome 

233 


Beckman Institute, University of Illinois at Urbana-Champaign, United States of America 

The fidelity of protein synthesis in the ribosome relies on the combined accuracy of three 

independent processes: charging of tRNAs by the corresponding aminoacyl-tRNA 

synthetases, selection of cognate aminoacyl-tRNA by the ribosome, and the recently 

discovered quality control by the ribosome detecting mismatches of the codon-anticodon 

interactions after peptidyl transfer. Using an all-atom structure of the ribosome in complex with 

tRNAs as well as cognate and noncognate mRNA codons, we investigate the mechanism of 

the quality control step employing equilibrium molecular dynamics and free energy methods. 

The simulations reveal the impact of the noncognate interactions on the ribosome structure 

and dynamics.



MIKHAIL SHCHEPETILNIKOV 

The role of TOR kinase in regulation of translation reinitiation events during 

CaMV infection 

Mikhail Shchepetilnikov, Lyubov Ryabova 

Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 

Strasbourg, France, France 

TOR kinase activation by growth factors and nutrients normally increases general level of 

translation. TOR is known to phosphorylate 4E-BPs, eIF4G, eEF2 and the S6K1 kinase. In 

plants, TOR is crucial for plant development, but role in regulation of translation requires further 

elucidation. We present data that TOR is a part of the plant translation initiation complex (PIC) 

and can pull-down eIF3, eIF2 and 40S ribosomal subunits. We study the alternative translation 

reinitiation mechanism used by Cauliflower mosaic virus (CaMV) for translation of polycistronic 

RNA activated by viral factor transactivator/ viroplasmin (TAV). TAV interacts with the host 

translation machinery via eIF3 and the 60S ribosomal subunit to allow polycistronic translation. 

It prevents loss of eIF3 from the translating ribosome during the elongation event. We found 

TAV interacts with TOR, and both can be immunoprecipitated from plants. The TOR N-terminal 

HEAT repeat domain is responsible for TAV binding. Using confocal microscopy of living plant 

cells we demonstrated co-localization between TOR and TAV fused to fluorescent proteins. 

Analysis of healthy and infected plants by IP methods suggested TOR is part of TAV/eIF3/40S 

translational complexes. Transient expression experiments in plant protoplasts revealed that 

TOR overexpression stimulates TAV-mediated polycistronic translation, while mutant lacking 

the active kinase domain was inactive. The downstream target of TOR in virus-induced 

transactivation seems to be S6K1. Indeed, in TAV-transgenic, CaMV infected, but not in 

healthy plants we detected a strong increase in the phosphorylation level of S6K1 at Thr389, 

TOR phosphorylation specific site. We speculate TAV binds TOR to modulate signaling to 

S6K1. This might lead to activation of the consecutive steps of translation reinitiation. At our 

knowledge this is the first example of direct interaction between virus and TOR. Analysis of 

plants with TOR knockdown will be presented. 

234

NARA SHIN 

Premature Termination Codon Containing mRNAs Are Translationally 

Repressed NMD-inhinition 

Yonsei Univ. College of Medicine, Korea, Republic of 

Nara Shin 1,2* , Kwon Tae You 1,2* , Hoguen Kim 1,2 

1 

Department of Pathology, Yonsei University College of Medicine, Seoul, Korea 

2 

Brain Korea 21, Project for Medical Science, Yonsei University 

* equal contributors 

235 


Quality control of expressed mRNA is crucial to regulation of gene expression. One of the 

quality control of mRNA expression is nonsense-mediated decay (NMD) which is a 

posttranscriptional mechanism that rapidly degrades mRNAs containing premature termination 

codon (PTC). For the NMD-resistant PTC containing mRNAs, the protein expression is 

repressed through a novel mechanism, nonsense-mediated translational repression (NMTR). A 

fundamental question is whether NMD and NMTR is a separate mechanism or NMTR is 

complementary mechanism for NMD. To address this question, we inhibited NMD in the 

NMD-sensitive PTC-containing beta-globin mRNAs, and evaluated the generation of truncated 

proteins. We found that PTC-containing beta-globin mRNAs are expressed after 

down-regulation of UPF1, however the abnormal protein is not generated. We demonstrated 

that the abnormal protein is not generated through translational repression using polysome 

fractionation assay. Finally, we demonstrated that translational repression is operated in the 

post-initiation step while NMD is operated in the initiation step. These findings indicate that 

NMD and NMTR are complementary mechanism targeting the PTC-containing mRNA from 

expression.



NIKOLAY SHIROKIKH 

Quantitative analysis of ribosome-mRNA complexes at different translation 

stages 

Elena Alkalaeva 1, Zhanna Afonina 2, Olga Alekhina 2, Lev Kisselev 2, Alexander Spirin 2, 

Nikolay Shirokikh 2, Konstantin Vassilenko 2 

1 Engelhardt Institute of Molecular Biology, Russian Federation 

2 Institute of Protein Research, Russian Federation 

Inhibition of primer extension by ribosome-mRNA complexes (toeprinting) is a proved and 

powerful method for studying mechanisms of mRNA translation by ribosomes. In this work we 

applied new toeprinting approach to detection and quantitation of different types of 

ribosome-mRNA complexes assembled in both, translation system reconstituted from purified 

components and non-fractionated cell lysates. The method is based on the use of fluorescently 

labeled DNA primers and capillary electrophoresis by standard instruments for sequencing and 

fragment analysis. We demonstrate that this approach is not merely fast and cost-effective but 

also brings a primer extension inhibition technique on a next level up. The electrophoretic 

pattern of primer extension reaction can be characterized with precision inaccessible for 

common toeprint analysis utilizing radioactive isotopes. We discuss optimization of reverse 

transcription reaction, detection and quantitation of ribosomal complexes on mRNA. We also 

point out some unique advantages of the new methodology, like a possibility to assay multiple 

sites of ribosomal complex assembly on mRNA in the same reaction mixture. 

236

RACHEL SIMMONDS 

237 


Gene specific translational control by an immunosuppressive mycobacterial 

virulence factor 

Rachel Simmonds 1, Anne Willis 2, Kirsti Hill 2, Pamela Small 3 

1 Imperial College, United Kingdom 


3 University of Tennessee, United States of America 

In bacterial infections, components of the cell wall elicit and immediate and strong innate 

immune response in cells such as monocytes and macrophages, causing inflammation and 

mobilising the adaptive immune response. In contrast, subcutaneous infection with 

Mycobacterium ulcerans (M.ulcerans) causes a debilitating disease known Buruli Ulcer, without 

any local or systemic inflammation. This immunosuppression is attributed to mycolactone, a 

polyketide toxin synthesised by the bacteria, but its mechanism of action is not known. 

We have found that mycolactone strongly and dose-dependently inhibits the production of 

inflammatory mediators without a concomitant effect on the steady state levels of mRNA 

transcripts, NF-kB/MAPK activation, or secretion. This suggests that mycolactone exerts its 

effect by translational control although the overall translation rate, as assessed by metabolic 

labelling, is not affected. Instead, mycolactone seems to inhibit the translation of a specific set 

of genes including TNFa, IL-6, IL-8, IL-10, IP-10, MCP-1, Cox-2 and Bcl-3; whereas the 

production of IkBa and actin are not affected. Mycolactone inhibits translation of these 

mediators in a broad range of cell types; indeed, the translation of an NF-kB-dependent 

luciferase reporter is also inhibited in primary human macrophages. 

We have investigated mycolactone’s effect on the known pathways controlling translation 

initiation, including the phosphorylation of eIF2a, eIF4E, eIF4E-BP1, the mTOR and PI3 kinase 

pathways, but these are not influenced. Polysome profiling studies of cells treated with 

mycolactone should give greater insights into the exact mechanism of translational control.



ANGELITA SIMONETTI 

Structural analysis of the translation initiation process 

Stefano Marzi 1, Angelita Simonetti 2, Marat Yusupov 2, Bruno Klaholz 2 


2 IGBMC Strasbourg, France 

In prokaryotes, three factors (IF1, IF2, IF3) kinetically assist the correct formation of the two 

ribosomal complexes that characterize the initiation step of translation, 30S (30SIC) and 70S 

initiation complex (70SIC). IF2 is involved in the fMet-tRNAfMet stabilization on the 30SIC and 

mediates subunit joining upon which its GTPase activity is stimulated.Recent cryo-EM 

reconstructions of the 30SIC complex with IF1 and IF21 has revealed how GTP-bound IF2 and 

fMet-tRNAfMet cooperate for their mutual stabilization and functional positioning on the 30S, 

enabling codon/anti-codon interaction in the ribosomal 30S P-site and favoring subunit joining. 

The comparison of this 30SIC structure with previously published 70SIC structures2,3, reveals 

how flexible is IF2, showing several conformational changes that accompanies the GTP 

hydrolysis, the release of its interaction with the initiator tRNA and its ribosomal relocation. 

Moreover, IF2 cryo-EM structures show different degrees of divergence from the only available 

crystal structure of an archaeal factor, IF2/eIF5B, that share 40% sequence identity with IF2. 

Specifically, a structural rearrangement of domains II and III seams to occur on the ribosome. 

High resolution structure of bacterial IF2 in combination with flexible dynamic fitting should 

provide a more detailed interpretation of cryo-EM maps necessary for a deeper understanding 

of the translation initiation process. 

Reference: 

1 Simonetti A. et al. (2008) Nature, 455: 416-420. 

2 Allen G.S. et al. (2005) Cell, 121: 703-712. 

3 Myasnikov A.G. et al. (2005) NSMB, 12: 1145-1149. 

238

MASAAKI SOKABE 

239 


Formation of human Multi-Factor Complex with purified components for EM 

structural analysis 

Masaaki Sokabe, John Hershey 

School of Medicine, University of California, Davis, United States of America 

Translation initiation is a rate-limiting step of protein synthesis, and thus is the important target 

for a number of regulation pathways in eukaryotes. Initiation process involves the binding of 

Met-tRNAi to 40S ribosome, recruitment of ribosome to 5’-end of an mRNA, scanning the 

downstream untranslated-region, recognition of the start codon, and following junction of 60S 

ribosome, in which at least 12 essential initiation factors are required. In yeast, eIF1, eIF3, eIF5 

and eIF2-GTP-Met-tRNAi can form multi-factor complex (MFC) prior to bind ribosome, which 

would be an important intermediate for Met-tRNAi delivery. Although the individual structures 

of MFC components have been solved by crystal/NMR (eIF1, eIF2 (archaea), and eIF5) and 

cryo-EM (eIF3) structural analyses, it is still unclear how these factors associate with each other 

and with ribosome/mRNA/tRNA to exert their functions in the course of initiation steps. The 

purpose of this study is to provide the structural basis of initiation complex assembly by 

cryo-/staining-EM analysis of MFC and 43S ribosome. We have purified human eIF2 and eIF3 

from HeLa cell extract, and also purified recombinant eIF1, eIF1A, eIF2beta, eIF2beta-NTD, 

eIF3c-NTD, eIF5, and eIF5-CTD. As most interactions among eIFs have been studied with 

yeast factors, we first tested whether human factors can interact with each other by pull-down 

and native-gel analyses. The result indicated the strong interactions among eIF1-eIF3 and 

eIF2/2beta-eIF5, and the weak interactions among eIF1-eIF5 and eIF5-eIF3/3c-NTD, mostly 

resembling those in yeast counterparts. Although no direct interactions among eIF1-eIF2 and 

eIF2-eIF3 were observed, eIF2-eIF1-eIF3 and eIF2-eIF5-eIF3 ternary complexes can stably be 

formed, suggesting that the formations of these ternary complexes are in cooperative manners. 

Finally, human MFC is stably formed in native-gel analysis even in a diluted condition (30nM). 

Sample preparation for EM analysis is in progress.



JOERG SOPPA 

Initiation and regulation of translation in halophilic Archaea 

Goethe-University, Frankfurt, Germany 

Translation of transcripts into proteins is a central process in expression of the genetic 

information. Regulation of translation plays important roles in development, stress response, 

and adaptation to new environments. Initiation of translation is rate limiting and therefore 

regulation takes place at this step. Four different mechanisms for translation initiation were 

known: 1) Shine Dalgarno (SD) sequence-dependent initiation, 2) initiation on leaderless 

transcripts, 3) the eukaryotic scanning mechanism, and 4) IRES-dependent initiation. Much 

less is known about translation initiation in Archaea, therefore we started to characterize 

translation initiation in haloarchaea. Determination of the 5’-ends of 40 transcripts and a 

genome-wide bioinformatic analysis revealed that the majority of transcripts is leaderless, and 

thus this mechanism, thought to be very seldom in bacteria, is the default pathway in 

haloarchaea. Surprisingly most transcripts with 5’-UTR were devoid of a SD sequence, 

excluding usage of the main bacterial pathway. In addition, it was excluded that a 

eukaryotic-like scanning mechanism is used for initiation on these transcripts. Therefore, a fifth 

and novel mechanism exists at least in haloarchaea, in addition to leaderless and 

SD-dependent initiation. 

Genome-wide ribosomal profiling was used to study growth phase-dependent translational 

regulation in two haloarchaeal species and it was revealed that 20% and 6%, respectively, of 

all genes were differentially regulated, showing that translational control is as common in 

haloarchaea as in eukaryotes. A reporter gene system was established that allows to study 

translational control in vivo. 5’- and 3’-UTRs alone are not sufficient, but both together are 

sufficient for translational control, indicating that they have to functionally interact in vivo. A UTR 

swap experiment revealed that the direction of regulation is encoded in the 3’-UTR. 

240

RICARDO SOTO RIFO 

241 


A deep comparison between HIV-1 and HIV-2 reveals strong differences in 

genomic RNA localization and their translational properties 

Ricardo Soto Rifo 1, Emiliano P. Ricci 1, Didier Decimo 1, Taran Limousin 1, Andrea Cimarelli 1, 

Theo Ohlmann 2 

1 Inserm U758 Ecole Normale Supérieure de Lyon, France 

2 INSERM-ENS de LYON, France 

HIV-1 infection is a major health problem being pandemic and responsible at least for a 90% of 

the total HIV cases worldwide. However, infection by the closely related lentivirus HIV-2 

strongly differs with those of HIV-1 being less pathogenic with an extremely lower progression 

to AIDS. It has been amply reported that differences in plasma viral load (or free circulating viral 

RNA) is an important factor that could explain the differences between both infections. This 

strongly suggests that after transcription and nuclear export, the cytoplasmic destiny of the 

full-length unspliced RNA, which is used as mRNA and genomic RNA, is essential for the 

success of infection. Here, we show that whereas the HIV-1 genomic RNA is located mainly at 

the cell plasma membrane indicating efficient viral production, the HIV-2 genomic RNA 

localizes in cytoplasmic foci containing the stress granules markers. In addition, we show that 

translation from the HIV-1 genomic RNA is 4 to 10-fold more efficient that those from the HIV-2 

genomic RNA indicating that the HIV-2 genomic RNA is encountered in a translationally 

repressed state. Furthermore, we were able to show that the HIV-2 5’-UTR confers a 

translational repression by blocking the entering of ribosomes to the 5’ cap structure. As a 

consequence, the HIV-2 genomic RNA is forced to use the IRES elements located in the Gag 

coding region. In sharp contrast, efficient protein production from HIV-1 corresponds to the 

ability of the genomic RNA to use a mix of cap-dependent and IRES-driven mechanisms for 

ribosome recruitment both orchestrated mainly by the viral 5’-UTR. Taking together, our results 

demonstrate that the HIV-1 and HIV-2 5’-UTR influence the mechanism and the efficiency by 

which the full-length unspliced genomic RNA is translated. As a consequence, protein 

synthesis strongly differs between HIV-1 and HIV-2 and represents a major determinant that 

may account for differences in viral pathogenesis.



JEFFREY SQUIRES 

Mapping 5-methylcytosine in RNA using bisulfite sequencing 

Marco Nousch 1, Jeffrey Squires 2, Catherine Suter 2, Preiss Thomas 2 

1 Max Planck Institute of Molecular Cell Biology and Genetics, Germany 

2 Victor Chang Cardiac Research Institute, Australia 

Modified ribonucleotides play important roles in RNA function and have been identified in 

multiple species including tRNAs, rRNAs, mRNAs, miRNAs, and other small RNAs. Among 

these, 5-methylcytosine (m5C) has been detected in rRNAs, tRNAs, and it was also reportedly 

present at very low level in mRNAs. Known and well studied as an epigenetic regulator of 

transcription in DNA, the prevalence and function of m5C in RNA is largely unexplored. 

Detection of m5C within DNA is readily carried out using bisulfite sequencing. The technique 

involves chemical sulfonation and deamination of cytosine residues (but not m5C) followed by 

alkaline desulfonation to uracil and nucleotide sequencing. Although bisulfite treatment for 

m5C mapping within RNA has rarely been used (1,2), we have been able to adapt the 

procedure for use with mammalian total RNA or mRNA samples. For method development we 

spiked cellular RNA samples with in vitro transcribed RNA cocktails as negative controls, while 

endogenous aspartyl-tRNA (an RNA with known m5C residues) was used as a positive 

control. After optimization of several experimental parameters, we were able to achieve 

>99.8% conversion, while we detect known m5C residues at ~96% success rate, as 

measured by conventional cloning and sequencing. We are now adjusting our approach for 

whole transcriptome cytosine methylation analysis using SOLiD 3 next generation 

sequencing technology. 

References 

1. Gu W, Hurto RL, Hopper AK, Grayhack EJ, Phizicky EM. Mol Cell Biol. 2005 

Sep;25(18):8191-201. 

2. Schaefer M, Pollex T, Hanna K, Lyko F. Nucleic Acids Res. 2009 Feb;37(2):e12. 

242

ABIGAIL STEVENSON 

243 


Intra-ribosome FRET and cryo-EM reveal conformational changes in the yeast 

43S ribosomal complex 

Abigail Stevenson 1, Pedro P. Juanes 1, John E.G. McCarthy 1, Luigi de Colibus 2, Robert 

Gilbert 2 

1 Manchester Interdisciplinary Biocentre, United Kingdom 


X-ray crystallography and cryo-electron microscopy have provided important insights into the 

molecular structures of prokaryotic ribosomes. In comparison, our understanding of the 

structural features of eukaryotic ribosomes is far more limited and particularly little is known 

about the dynamics of conformational changes in the 40S-based ribonucleoprotein 

complexes. Here, we report progress towards characterization of the conformational states of 

the 40S ribosomal subunit and of its complexes using a combination of cryo-EM and 

fluorescence resonance energy transfer (FRET) methods. These provide striking evidence of 

the highly dynamic nature of the ribosome and direct correlations between factor binding and 

conformational events. We have purified S.cerevisiae 40S subunits plus initiation factors 

involved in the generation of the mRNA-recruitment-competent 43S complex. We present high 

resolution cryo-EM reconstructions of the 43S that are much improved over those previously 

published, though entirely in agreement with them. They reveal with much-enhanced detail 

how eIF binding induces mobility in the head and platform and reconfigures the 

head-platform-body relationship. The head moves down over the solvent face of the small 

subunit, and the platform is dramatically changed in orientation, in ways enabled by the 

underlying RNA skeleton of the subunit. The dynamic nature of the ribosome was further 

investigated using fluorescence techniques. We have tagged 40S subunits at different pairs of 

sites using GFP and the tetracysteine motif (TCM) that binds fluorescent bi-arsenical dyes, 

creating double-tagged 40S subunits. Using FRET, we can monitor intra-subunit movement 

between the head and body during different phases of translation initiation. For the first time, 

detailed structural data are combined with conformational dynamics results to generate a 

picture of the mobility of the eukaryotic ribosome structure.



NADINE STÖHR 

ZBP1 controls mRNA turnover during stress independent of stress granules 

Nadine Stöhr, Stefan Hüttelmaier 

University of Halle, Germany 

Cellular stress leeds to a reversible translational arrest resulting in a sequestering of bulk mRNA 

with RNA-binding proteins like ZBP1 in stress granules (SGs). It is presumed that bulk mRNA is 

stored in a translational dormant manner in SGs as there is no evidence for mRNA degradation 

within these foci. However, the role of SGs in mediating mRNA stabilization during cellular 

stress remains elusive. In previous studies we demonstrated that ZBP1, a protein that 

regulates turnover or translation of its target mRNAs in unperturbed cells, exclusively stabilizes 

these transcripts during stress. This stabilization of target mRNAs during stress is selective and 

facilitated by ZBP1 associating with the same cis-determinants required to direct transcript fate 

in non-stressed cells. Irrespective of stress the protein is also associated with the same 

RNA-binding proteins in cytoplasmic mRNPs. Some of these mRNPs apparently aggregated to 

SGs during stress by associating with TIA-proteins and/or G3BP. Interestingly, when 

SG-formation was prevented, global RNA stability remained unaffected, whereas stabilization 

of mRNAs by ZBP1 was still preserved. These findings indicate that the stabilization of ZBP1 

target mRNAs is facilitated by ZBP1-containing mRNPs, whereas SG-formation is not 

obligatory for mRNA stabilization during stress. 

244

SURESH SUSMITHA 

245 


Characterization of ribosomal/ribosomal associated proteins in S.cerevisiae 

University of Maryland Baltimore County, United States of America 

A screen identified a set of proteins that restricted Ty1 transposition (Ty1 ‘restriction’ genes) 

and another that helped in Ty1 transposition (Ty1 ‘helper’ genes) in S.cerevisiae. The Ty1 

helper genes included some genes that encoded the large subunit and small subunit ribosomal 

proteins. The list also included other genes that are associated with either translation or 

ribosome biogenesis. Strains harboring deletion of the above said genes were assayed for Ty1 

programmed frameshift activity. Interestingly one of the ribosomal proteins that show 

decreased +1 frameshifting (but increased transposition) is ASC1, homolog of mammalian 

RACK1, a signaling protein in the glucose response pathway. ASC1 interacts with the 

ribosome and Gpa2 (protein involved in the glucose response pathway) using the same 

interacting surface. The main goal of my project is to further test the deletion strains for rRNA 

processing defects, increased sensitivity to antibiotics and budding defects. We will also further 

extend our analysis on ASC1 protein by doing a random mutagenesis and isolate mutants that 

affect the glucose response pathway without affecting its ribosome function.



SYLWIA SZCZEPANIAK 

Cap analog modified enzymatically stable affinity resins – a new tool for the 

analysis of cap binding proteins 

Edward Darzynkiewicz 1, Jacek Jemielity 1, Joanna Zuberek 1, Sylwia Szczepaniak 2, Andrzej 

Dziembowski 2, Joanna Kufel 2 

1 Departament of Biophysics, Institute of Experimental Physics, Faculty of Physics, Warsaw 

University, Poland 

2 Department of Genetics, Warsaw University, Poland 

The 5’ cap of eukaryotic mRNAs plays a crucial role in regulation of gene expression. It 

determines mRNA stability and participates in several cellular processes, above of all 

translation initiation. Cap recognition by specific proteins is essential for its biological function. 

There are two major cap-binding complexes: primarily nuclear CBC and mainly cytoplasmic 

eIF4F. Another important general modulator of cap-dependent processes is the scavenger 

mRNA decapping enzyme (DcpS). Affinity chromatography is a useful method for purifying 

proteins responsible for specific activities in the case when their binding characteristics are 

known. m7GTP-Sepharose is routinely used for isolation of cap-binding proteins, but it may be 

inefficient for proteins, such as DcpS, interacting not only with the 7-methylguanosine but also 

with the second base of the cap. In addition, DcpS is able to cleave off the m7GTP ligand, 

which will prevent its purification and reduce resin capacity. Here we report the synthesis of 

methylene-modified cap analog Sepharoses, particularly m7GpCH2ppA-Sepharose that is the 

first affinity resin resistant to hydrolysis by DcpS and therefore suitable for its purification. We 

synthesized three types of resins: m7GpppA-Sepharose, and two enzymatically stable 

matrices: m7GpCH2pp-Sepharose and m7GpCH2ppA-Sepharose. Biochemical tests showed 

that all affinity resin specifically bind mouse eIF4E(28-217) protein with high binding capacity. 

To test the applicability of new matrices affinity purification of cap-binding proteins from yeast 

and plant extracts were carried out. Mass spectrometry of eluted fractions confirmed the 

presence of all expected cap binding proteins including DcpS in the case of m7GpCH2pp- and 

m7GpCH2ppA-Sepharoses. These results show that synthesized modified cap analog 

Sepharoses can be effectively utilized for identification of new cap-binding proteins from 

different organisms. 

246

ABDESSAMAD TAHIRI-ALAOUI 

247 


The 5' Leader of an immediate-early transcript from Marek's Disease Virus 

contains intronic IRES with allosteric properties 

Institute for Animal Health, United Kingdom 

Abdessamad Tahiri-Alaoui 1 , Daiki Matsuda 2 , Hongtao Xu 1 , Panopoulos Panagiotis 2 , Luke 

Burman 2 , Luke S. Lambeth 1 , Lawrence Petherbridge 1 , William James 3 , Vincent Mauro 2 , 

Venugopal Nair 1 

1 Institute for Animal Health, Division of Microbiology, Compton, Berkshire RG20 7NN, UK 

2 Department of Neurobiology, The Scripps Research Institute and The Skaggs Institute for 

Chemical Biology, La Jolla, California 92037, USA 

3 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 

3RE, UK 

We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' 

leader (5L) of the bicistronic mRNA that encodes the pp14 and RLORF9 proteins from Marek’s 

disease virus serotype-1 (MDV-1). The transcript is a member of the 1.8-kb family of transcripts 

that is expressed as an immediate-early gene. The 5L IRES is contained within a sequence that 

is retained in a subset of the mature bicistronic transcripts as a result of alternative splicing or 

alternative promoter usage. Real time reverse transcription (RT) quantitative PCR (qPCR) 

indicates that the mRNA variant with the 5L IRES is more abundant in MDV-infected and 

transformed cells than the mRNA variant lacking the 5L IRES. The different mRNAs give rise to 

two variants of the pp14 protein that differ in their N-terminal sequences. A common feature to 

all 1.8-kb family of transcripts is the presence of an intercistronic (ICR) IRES that we have 

previously shown to control the translation of the second open reading frame (RLORF9). 

Investigation of the interaction between the two IRESes revealed functional synergy between 

the 5L IRES and the ICR IRES within the same bicistronic RNA. In analogy with allosteric 

models in proteins we propose IRES allostery to describe such a novel phenomenon. The 

functional implications of our findings are discussed in relation to viral pathogenesis and 

translational control.



JUDY TELLAM 

Regulation of Protein Translation through mRNA Structure Influences MHC 

Class I Loading and T cell Recognition 

Tscharke David 1, Vuocolo Tony 2, Judy Tellam 3, Corey Smith 3, Rist Michael 3, Webb 

Natasha 3, Cooper Leanne 3, Connolly Geoff 3, Khanna Rajiv 3 

1 Australian National University, Australia 

2 Commonwealth Scientific Industrial Research Organization, Australia 

3 Queensland Institute of Medical Research, Australia 

Many viruses avoid immune surveillance during latent infection through reduction in the 

synthesis of virally encoded proteins. Although antigen presentation is critically dependent on 

the level of viral protein synthesis, the precise mechanism(s) used to regulate the generation of 

antigenic peptide precursors remains elusive. Here we demonstrate that a purine overloaded 

virally encoded mRNA lacking secondary structure significantly impacts on the efficiency of 

protein translation and prevents endogenous antigen presentation. Reducing this purine-bias 

through the generation of constructs expressing codon-modified sequences, whilst maintaining 

the encoded protein sequence, increased secondary structure of the corresponding mRNA 

and dramatically enhanced selfsynthesis of the viral protein. As a consequence, a higher 

number of HLA-peptide complexes were detected on the surface of cells expressing this viral 

protein. Furthermore, these cells were more efficiently recognized by virus-specific T-cells 

compared to those expressing the same antigen expressed by a purine-biased mRNA. These 

findings delineate a novel mechanism by which viruses regulate self-synthesis of proteins and 

offer an effective strategy to evade CD8+ T-cell mediated immune regulation. 

248

CLAUDIA TEMME 

The Drosophila CCR4 NOT deadenylase: composition and function 

Claudia Temme, Elmar Wahle, Lianbing Zhang 


249 


Two general pathways of mRNA decay have been characterized in yeast. Both start with the 

deadenylation. These pathways are believed to be conserved in higher eukaryotes, which 

could be confirmed for Drosophila (5). The CCR4∙NOT complex is the main deadenylase in 

yeast (1) and its composition is well studied (2). Most of its subunits are also present in 

Drosophila (3). 

Co-immunoprecipitation experiments with antibodies against the different subunits from either 

Drosophila embryo - or S2 cell extract show association of most of the subunits. The complex 

is located predominantly in the cytoplasm. Knock down of single subunits by RNAi impairs 

deadenylation to different levels. The knock down of CAF1, one of the exonuclease-motif 

containing subunits of the CCR4 NOT complex, shows the strongest effect. Surprisingly, a 

severe reduction of CCR4, the major deadenylase activity in the yeast complex (1), has very 

little effect on deadenylation in S2 cells or in mutant flies (3). One reason may be that the 

activity of the residual proteins is sufficient to confer deadenylation. Alternatively, the CCR4 

homologues angel, nocturnin and d3635, present only in higher eukaryotes (4), might be able 

to replace CCR4 in the complex. To test this hypothesis, we expressed CCR4 and its 

homologues as FLAG-tagged proteins in S2 cells and performed immunoprecipitation 

experiments: Only FLAG-CCR4 was able to precipitate CAF1 from Schneider cell extract, 

suggesting that its homologues do not associate with the CCR4 NOT complex. 

(1) Tucker, M., et al (2001) Cell 104, 377-386 

(2) Chen, J., et al. (2001) J. Mol. Biol., 314, 683-694 

(3) Temme, C., et al. (2004) <strong>EMBO</strong> J. 23, 2862-2871 

(4) Dupressoir, A., et al. (2001) BMC Genomics. 2(1):9 

(5) Boenisch, C., et al. (2007) J Biol Chem. 282(30):21818-28



ILYA TERENIN 

Delay in eIF5-mediated hydrolysis of eIF2-bound GTP regulates start codon 

selection during translation initiation in mammals 

Ilya Terenin, Sergey Dmitriev, Dmitry Andreev, Ivan Shatsky 

Moscow State University, Russian Federation 

According to the generally accepted view, AUG recognition by scanning 43S complexes sets 

the final point in a process of start codon selection during eukaryotic translation initiation. On 

mRNA containing two or more AUG codons, the establishment of the stable codon-anticidon 

base paring traps the ribosome at the first AUG in a good context and therefore 

unambiguously determines the point of protein synthesis initiation, while later stages, such as 

eIF2-bound GTO hydrolysis and 60S subunit joining, have not been reported to contribute to 

selection between alternative AUG triplets in mammals. Here, using translation initiation 

reconstitution approach combined with kinetic toeprinting on a set of different mRNAs with two 

or more closely spaced AUG codons, we show that after the 48S complex formation at the 

first AUG codon in a good context under conditions when GTP hydrolysis is impaired, the 40S 

ribosome is able to resume scanning and slides along the mRNA to closely positioned 

downstream initiation codons. In contrast to conventional leaky scanning, such sliding only 

occurs after a rather long pause at the first AUG codon. Thus, recognition of a “good” AUG by 

the anticodon of initiator tRNA per se does not irreversibly lead to translation initiation from this 

particular codon. In contrast, eIF5-induced GTP hydrolysis leading to eIF2-GDP dissociation 

traps the 48S complex, and such a complex lacking eIF2 is further stabilized by eIF5B and 60S 

joining. This suggests a novel mechanism of translation regulation under conditions when 

activities of subunit joining factors are affected. Indeed, we were able to show that changes of 

eIF5 concentration in cell-free systems do significantly affect the ratio of polypeptides 

synthesized from a single mRNA containing two overlapping ORFs, and also regulate 

translation efficiency of mRNAs with uAUG(s). The physiological significance of these 

observations will be discussed. 

250

ROLF THERMANN 

251 


Drosophila miR2 primarily targets the m7GpppN cap structure for translational 

repression 

Joanna Kowalska 1, Jacek Jemielity 1, Edward Darzynkiewicz 1, Rolf Thermann 2, Agnieszka 

Zdanowicz 2, Matthias W. Hentze 2, Thomas Preiss 3 

1 Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of 

Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland, Poland 

2 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany 

3 Molecular Genetics Division, Victor Chang Cardiac Research Institute, 405 Liverpool 

Street, Darlinghurst (Sydney) NSW 2010, Australia 

Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation 

represents a fundamental challenge in RNA biology. Using a validated cell-free system from 

Drosophila embryos, we have investigated how miR2 inhibits translation initiation. We screened 

a library of chemical m7GpppN cap structure analogs to select variants that are inert towards 

general cap-dependent translation. We then used these “neutral” cap variants and identified 

two defined modifications of the triphosphate backbone that specifically augment 

miRNA-mediated inhibition of translation initiation both in vitro and in vivo without affecting 

mRNA stability. Additionally, we observe miR2-induced mRNA deadenylation in vitro. Kinetic 

dissection of translational repression and deadenylation shows that both processes can 

proceed largely independently, with the establishment of the repressed state involving a slow 

step. Our data define the m7GpppN cap structure as a primary target for translational inhibition 

that can be augmented by deadenylation.



MARY K. THOMPSON 

Ribosome-profiling of a RACK1 ribosome-binding defective mutant in response 

to Calcofluor-white induced stress 

Mary K. Thompson, Wendy Gilbert 

Massachusetts Institute of Technology, United States of America 

The receptor for activated C-kinase (RACK1) has been extensively studied for its role as an 

anchor for protein kinase C (PKC) and is an essential component of many signaling pathways. 

In addition to this role, it is a stochiometric component of eukaryotic 40S ribosomal subunits. In 

yeast, RACK1 ribosome-binding defective mutants are viable and sensitive to Calcofluor-white 

(CFW), a drug that induces cell wall stress and activates the PKC-dependent cell integrity 

signaling pathway. Recent genomic studies showed that CFW induces only minor changes in 

the total RNA population while selectively affecting the association of several transcripts with 

polysomes. The CFW-sensitive phenotype of RACK1 ribosome-binding mutants suggests the 

intriguing possibility that RACK1 regulates the association of specific transcripts with the 

ribosome and thus provides a mechanism for the integration of signaling pathways with the 

translation machinery. Such a mechanism could allow direct communication of the cellular 

state to guide appropriate protein synthesis, which could provide both a temporal and spatial 

advantage over a transcriptional response. To probe this mechanism, we are using a recently 

developed ribosome-profiling technique involving deep sequencing of ribosome-protected 

RNA fragments to permit quantitative, genome-wide analysis of translation. This analysis will 

pave the way for future investigation of signaling integration via RACK1 at the site of protein 

synthesis. 

252

SUNNIE THOMPSON 

A ribosomal protein that is essential for IRES-mediated translation 

Sunnie Thompson, Landry Dori, Hertz Marla 

University of Alabama at Birmingham, United States of America 

253 


The vast majority mRNAs are translated using a cap-dependent mechanism of translation. 

However, 5% to 10% of messages initiate translation using a cap-independent mechanism 

that is not well understood. These mRNAs contain an internal ribosome entry site (IRES) 

located in the 5' UTR and are able to initiate translation independent of a cap structure. The 

cricket paralysis virus (CrPV) intergenic region IRES (IGR IRES) functions robustly in yeast and 

does not require any initiation factors. We generated a strain lacking all copies of the ribosomal 

protein S25 (Rps25) and demonstrated that Rps25p is essential for IGR IRES-mediated 

translation while cap-dependent translation is unaffected. 40S ribosomal subunits lacking 

Rps25 protein are unable to bind to the IGR IRES in vitro. We confirmed that Rps25p is also 

necessary for CrPV IGR IRES-mediated translation in mammalian cells by using siRNA to 

knockdown Rps25. To determine whether ribosomes lacking Rps25 protein are deficient in 

any other functions we assayed for effects on global translation, ribosome biogenesis, 

readthrough and frame-shifting. Surprisingly, we found that the rps25aΔ/rps25bΔ yeast strain 

had no affect an global translation, and only a mild defect in ribosome biogenesis, suppresses 

readthrough two to five-fold greater than observed in the wild-type strain and demonstrated a 

minor increase in +1 programmed frame-shifting. This work is highly significant because it is 

the first demonstration of a ribosomal protein that is specifically required for IRES-mediated 

translation. Taken together, our findings are beginning to provide us with a model of functional 

IRES interactions with the ribosome and suggest that there maybe specialized ribosomes for 

IRES-mediated translation in the cell.



JULIANO TOLEDO 

Leishmania mutants overexpressing the Spliced Leader RNA present an 

altered pattern of gene expression and are unable to cause infection in vivo 

Serge Cloutier 1, Fernando M Dossin 2, Sergio Schenkman 2, Tiago R Ferreira 3, Tânia PA 

Defina 3, Simone A Antoniazi 3, Angela K Cruz 3, Douglas J Lamont 4, Kenneth A Beattie 4, 

Barbara Papadopoulou 5, Juliano Toledo 6 

1 Research Centre in Infectious Diseases of CHUL, Laval University, Canada 

2 Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São 

Paulo, Brazil 

3 Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil 

4 Fingerprints Proteomics Facility, School of Life Sciences – University of Dundee, United 

Kingdom 

5 Research Centre in Infectious Diseases of CHUL, Laval University, Canada 

6 Universidade de São Paulo, Brazil 

Leishmania parasites are etiological agents of leishmaniasis. These microorganisms exhibit 

complex regulatory mechanisms of gene expression, which are not yet fully understood. RNA 

polymerase II promoters for protein coding genes have never been detected and modulation of 

gene expression occurs at the post-transcriptional level. DNA transcription generates long 

primary polycistronic RNAs, which are resolved into mature mRNAs by trans-splicing reaction 

coupled with polyadenylation. This bimolecular process transfers a 39nt sequence known as 

Spliced Leader (SL RNA) to the 5’ end of each mRNA in the polycistronic primary transcript. 

We have demonstrated by in vivo infections that parasites overexpressing SL RNA are unable 

to establish infection in animal models. Transcriptome and proteome analyses of this mutant 

revealed abnormal pattern of expression of 110 genes related to stress response, 

cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, transcription 

and processing machineries, and to post-trancriptional regulation. The polysome profile 

analysis and evaluation of cellular protein aggregates indicates that protein translation in the SL 

overexpressor is modified compared to a control line. Immunoblots and enzymatic assays 

revealed that the ubiquitin-proteasome system and the activity of cysteine and 

metalloproteases are altered in Leishmania SL RNA mutants. Morphological changes observed 

are suggestive that a subcellular structure known as MVT-lysosome is involved in regulation of 

protein levels in the mutant. The observed changes indicate the parasite effort to maintain cell 

homeostasis. Due to the SL RNA central role in mRNA maturation we propose that the 

imbalance generated by the SL RNA surplus on the Leishmania metabolism might interfere 

with a fine-tuned gene expression control necessary for the parasite multiplication in the 

mammalian host. 

Supported by FAPESP, CNPq and CBIE. 

254

LEONARDO TRABUCO 

255 


Computational studies of regulatory nascent chain recognition by the ribosome 

Christopher B. Harrison 1, Eduard Schreiner 1, Leonardo Trabuco 2, Klaus Schulten 3, 

Christophe Chipot 4 

1 Beckman Institute, University of Illinois at Urbana Champaign, United States of America 

2 Center for Biophysics and Computational Biology and Beckman Institute, University of 

Illinois at Urbana Champaign, United States of America 

3 Department of Physics and Beckman Institute, University of Illinois at Urbana-Champaign, 


4 Nancy Université, France 

The role of the ribosomal exit tunnel in translational control has been increasingly realized. 

Regulatory nascent chains interact with the exit tunnel and modulate their own translation. 

Extensive molecular dynamics simulations of the regulatory nascent chain TnaC inside the exit 

tunnel were performed for an aggregate time of 1.8 microseconds. The simulations, combined 

with bioinformatic analyses, reveal atomic-detail interactions that explain the role of critical 

residues in the nascent chain and ribosomal elements. Free energy calculations were also 

conducted to investigate a proposed mechanism for nascent chain-mediated translational 

arrest. Specifically, we calculated the free energy corresponding to a conformational change of 

ribosomal protein L22 for arrest-suppressing mutants with respect to the wild-type. Altogether, 

our results show similarities and differences in the action mode of TnaC and SecM, two 

regulatory nascent chains that induce translational arrest.



BECKY TSAI 

Identifying LEF1 IRES Protein Complexes by Mass Spectrometry 

Xiaorong Wang 1, Becky Tsai 2, Marian Waterman 2, Lan Huang 2 

1 University of California, Ca, United States of America 

2 University of California, Irvine, United States of America 

RNA-protein complexes underlie processes of post-transcriptional regulation. The dynamic 

nature of ribonucleoprotein complexes has made it challenging to identify components and 

define steps in RNA processes. We developed a strategy to characterize proteins bound to the 

IRES (internal ribosome entry site) motif on the LEF1 mRNA. IRESes are located in lengthy 5’ 

untranslated (UTR) regions of several eukaryotic mRNAs and provide an alternative mechanism 

to recruit translation factors and ribosomes. Unlike most mRNAs with short 5’UTRs, the LEF1 

IRES is a 1.2kb structured region which impedes ribosome scanning initiated at the mRNA 

cap. Instead, ribosomes are recruited through IRES binding proteins that have not been 

identified. New strategies are required to identify these IRES binding proteins bound to RNA. 

Due to its high affinity for a specific stem-loop structure, the MS2 bacteriophage coat protein 

was HB-tagged to affinity purify in vivo RNA-protein complexes. The target RNA is engineered 

with MS2 stem-loops and co-expressed with HB-tagged MS2 coat protein. Affinity purification 

was achieved using magnetic streptavidin beads and TEV cleavage elution for rapid isolation of 

the biotinylated coat protein and target IRES-protein complexes assembled in vivo. 

SILAC-based quantitative mass spectrometry was employed to specifically identify IRES 

interacting proteins. Our aim is to develop a strategy for isolation of bona fide in vivo 

assembled IRES-protein complexes for LC MS/MS identification. We identified ~50 proteins 

that were reproducibly enriched on the target RNA. We isolated several known and putative 

LEF1 IRES trans-acting factors. These preliminary results suggest that IRES motifs may not be 

regulated by a subset of unique factors but rather an assortment of canonical RNA regulatory 

factors. The method reported provides a versatile approach which can be applied to any 

RNA-protein interaction and is highly adaptable to additional techniques. 

256

JOSEPH TA-CHIEN TSENG 

Translational up-regulation of Aurora-A in EGFR-overexpressed cancer 

Chien-Hsien Lai, Joseph Ta-Chien Tseng, Liang-Yi Hung 

National Cheng-Kung University, Taiwan 

257 


Abnormal expression of Aurora-A and EGFR is observed in different kinds of cancer and 

associated with poor prognosis in cancer patients. However, the relationship between 

Aurora-A and EGFR in tumor development was not clear. In previously report (Nucleic Acids 

Res, 2008, 36:4337-4351), we found EGFR translocates to nucleic to activate Aurora-A 

expression after EGF treatment in EGFR-overexpressed cells. However, we also observed that 

not all the EGFR-overexpressed cells have the nuclear EGFR pathway to mediate the Aurora-A 

expression. In this study, we demonstrated that EGF signaling increased the Aurora-A protein 

expression in EGFR-overexpressed colorectal cancer cell lines via increasing the translational 

efficiency. In addition, the overexpression of EGFR was also associated with higher expression 

of Aurora-A in clinical colorectal samples. Activation of the PI3K/Akt/mTOR and MEK/ERK 

pathways mediated the effect of EGF induced translational up-regulation. Besides, only the 

splicing variants containing exon 2 of Aurora-A mRNA showed increased interaction with the 

translational complex to synthesize Aurora-A protein under EGF stimulus. Besides, the exon 2 

containing splicing variants were the major Aurora-A splicing form expressed in human 

colorectal cancers. Taken together, our results propose a novel regulatory mechanism for the 

abnormal expression of Aurora-A in EGFR-overexpressed cancers, and highlight the 

importance of alternative 5’-UTR splicing variants in regulating Aurora-A expression. 

Furthermore, the specific expression of exon 2 containing splicing variants in cancer tissues 

may serve as a potential target for cancer therapy in the future.



TAMIR TULLER 

A universal translation efficiency profile of proteins 

Kalin Vestsigian 1, Tamir Tuller 2, Asaf Carmi 3, Sivan Navon 3, Itay Furman 3, Yitzhak Pilpel 3 

1 Harvard Medical School, United States of America 

2 Tel-Aviv University, Israel 

3 Weizmann Institute of Science, Israel 

We report a universally conserved profile of translation efficiency along mRNAs based on 

computing the adaptation between coding sequences and the tRNA pool. In this profile the 

first ~30-50 codons of genes show a preference towards rare tRNAs, and are thus deduced to 

be translated with low efficiency, while, mostly in eukaryotes, the last ~50 codons show 

highest efficiency. The profile predicts well position-dependent ribosomal density along yeast 

genes – areas of low translation efficiency correspond to high density, indicating that 

translation speed, and as a consequence, ribosomal density, are encoded in codon-tRNA 

adaptation. Although both the tRNA pools and codon preferences change across species, 

their co-evolution appears to have conserved the universal profile, indicating its adaptiveness. 

Our model suggests that the observed efficiency profile is optimal in that it minimizes ribosomal 

traffic jamming hence minimizing consumption of free ribosomes from the cellular pool. 

258

NILGUN TUMER 

259 


Ricin A chain interacts with isolated ribosomal stalk in a single step binding 

model 

Przemyslaw Grela 1, Marek Tchorzewski 1, Nilgun Tumer 2, Xiao-Ping Li 2 

1 Maria Curie-Sklodowska University, Poland 

2 Rutgers University, United States of America 

Ricin depurinates the sarcin/ricin loop (SRL) and inhibits protein synthesis. Ricin A chain (RTA) 

interacts with P1 and P2 proteins of the ribosomal stalk to depurinate the SRL. We examined 

the interaction of RTA with yeast ribosomes using surface plasmon resonance (SPR) and 

demonstrated that this interaction did not fit a 1:1 binding model, but was characterized by a 

two-step binding model. During the first step, the negatively charged ribosomes interact with 

the positively charged C-terminus of RTA via nonspecific electrostatic interactions to 

concentrate the RTA molecules on the surface of the ribosomes. In the second step, the 

ribosomal stalk interacts with the C-terminus of RTA via more specific electrostatic interactions 

and delivers the RTA to the SRL. In this study we used SPR to investigate the interaction of 

RTA with the isolated native stalk pentamer from yeast. We showed that RTA interacts with 

the isolated stalk via a 1:1 binding model, consistent with our two step interaction model with 

intact ribosomes. This interaction occurred through the C-terminus of RTA. However, 

RTA-ribosomal stalk interaction was not as sensitive to increased concentration of salt. The 

dissociation rate of RTA from the isolated stalk was much slower than the dissociation rate 

from intact ribosomes, suggesting that the electrostatic interactions with the SRL may 

contribute to the dissociation of RTA from the stalk on intact ribosomes. We predict that the 

binding model proposed here may be more broadly applicable to the interaction of the 

ribosomal stalk with the elongation factors.



VARSHNEY UMESH 

Impact of rRNA methylations on ribosome recycling and fidelity of initiation in 

Escherichia coli 

Varshney Umesh 1, Seshadri Anuradha 1, Weber Michael H. W. 2 

1 Indian Institute of Science, Bangalore, India 

2 Rechenkraft.net e.V., Chemnitzer Str. 33, D-35039 Marburg, Germany 

We will discuss that RRF contributes to fidelity of initiator tRNA selection on the ribosome, and 

together with our earlier data propose that RRF plays a crucial role during all the steps of 

protein synthesis in Escherichia coli. 

260

LEOS VALASEK 

261 


The indispensable N-terminal half of eIF3j co-operates with its structurally 

conserved binding partner eIF3b-RRM and eIF1A in stringent AUG selection 

Leos Valasek 1, Susan Wagner 2, Anna Herrmannova 2, Martina Janoskova 2, Edit Rutkai 2, 

Latifa Elantak 2, Peter Lukavsky 2 

1 Institute of Microbiology, AS CR, Czech Republic 

2 MRC Laboratory of Molecular Biology, United Kingdom 

Despite the recent progress in our understanding of the numerous functions of eukaryotic 

translation initiation factor 3, there is still only little known on the molecular level. Using NMR 

spectroscopy, we determined the first solution structure of an interaction between eIF3 

subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal 

acidic domain (NTA) is held in the helix α1 – loop L5 hydrophobic pocket of the human 

eIF3b-RRM. Mutating the corresponding “pocket” residues in its yeast orthologue reduces 

cellular growth rate and affects 40S-occupancy of eIF3. Furthermore, we show that the 

N-terminal half (NTD) of eIF3j is indispensable and sufficient for wild growth of yeast cells and 

retains significant binding affinity towards the 40S ribosome. Strikingly, we also demonstrate 

that deletion of either eIF3j or its NTD only, or mutating the key tryptophan residue produces a 

severe leaky scanning defect indicative of a deregulation of the start codon selection process. 

Since this defect was found partially suppressible by overexpressed eIF1A and also observed 

with a specific eIF3b-RRM mutant, we propose that eIF3j closely co-operates with eIF3b-RRM 

and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation 

required for stringent AUG recognition.



NORA VAZQUEZ-LASLOP 

Regulation of translation by the nascent peptide 

Nora Vazquez-Laslop, Haripriya Ramu, Alexander Mankin 

University of Illinois at Chicago, United States of America 

Expression of several bacterial and eukaryotic genes is controlled by ribosome stalling which 

depends on interaction of the ribosome with the nascent peptide. In bacteria, regulation of 

expression of some inducible antibiotic resistance genes, including ermC, depends on 

formation of a stalled ribosomal complex (SRC) at the regulatory ORF located upstream from 

the resistance cistron. SRC formation is controlled by the presence of the inducing antibiotic 

and specific interactions of the nascent peptide with the elements of the ribosome exit tunnel. 

By using genetic and biochemical analysis, we discovered that a universally conserved and 

posttranscriptionally-modified 23S rRNA residue A2503, located in the exit tunnel, as well as 

the previously identified residue A2062, are critical for sensing and responding to the ermCL 

regulatory nascent peptide. Both A2503 and A2062 are structurally connected to the peptidyl 

transferase center thus providing a straightforward route for the signal transduction between 

the exit tunnel and the catalytic center of the ribosome. Mutational analysis of the stalling 

peptides and biochemical analysis of various SRCs suggest that the presence of specific 

nascent peptides in the ribosome exit tunnel direct alterations in the ribosomal A site which 

prevent peptide bond formation with a specific range of aminoacyl-tRNA substrates. 

Comparison of the inducing effects of different antibiotics indicates that recognition of specific 

elements of the drug structure by the ribosome is required for the SRC formation. Analysis of 

the ribosome stalling at regulatory ORFs of different inducible antibiotic resistance genes 

shows that various nascent peptide sequences can direct formation of the SRC. This result 

indicates that broad range of functional interactions between the ribosome and the nascent 

peptide may lead to formation of SRC and suggests that this type of gene regulation may have 

a general role in control of gene expression in the cell. 

262

BETHANY VEO 

Identification of potential ITAFS that regulate the TAU IRES 

Les Krushel, Bethany Veo 

1 University of Colorado Medical School, United States of America 

263 


Neurofibrillary tangles are a pathological phenotype in Alzheimer’s disease (AD) and are caused 

by the hyperphosphorylation of the microtubule associated protein tau. The accumulation of 

NFTs is associated with neuronal cell loss and cognitive decline. Previous studies have shown 

that reducing tau expression can partially restore cognitive deficits observed in animal models 

of AD. In light of this data a new emphasis has been placed on reducing tau protein levels in 

AD. In order to alter protein expression of tau we first examined a major step in protein 

expression, translation initiation. The primary mechanism of eukaryotic translation initiation is 

dependent on the recognition of a 7-methyl guanosine cap structure by eIF4E and the 

subsequent recruitment of canonical initiation factors. However, an alternative method of 

initiating translation occurs independently of the cap structure through an internal ribosomal 

entry site (IRES). Indeed, we have reported recently that translation initiation of tau mRNA can 

occur through an IRES (Veo and Krushel JAD 2009). Deletion analysis of the tau 5’ leader 

shows that the majority of the sequence is necessary for IRES function. 

IRES-dependent translation of eukaryotic mRNAs requires both canonical as well as 

non-canonical initiation proteins called IRES trans-acting factors (ITAFs). These factors are 

predicted to act as chaperones and alter RNA secondary structure making it more accessible 

to the ribosome, and/or serve as a factor to recruit the ribosome. Our examination of the tau 

5’ leader has revealed binding sites for the well-characterized ITAF polypyrimidine-tract binding 

protein (PTB) with a Kd ranging from 29 to 260 nM. Further analysis of the mechanism by 

which PTB may act on the tau IRES as well as identifying other potential ITAFs including the 

neural PTB isoform is currently ongoing.



GABRIELLA VIERO 

THE SHAPES OF NATIVE MAMMALIAN POLYSOMES 

Gabriella Viero 1, Lorenzo Lunelli 2, Cecilia Pederzolli 2, Natalia Arseni 3, Alessandro 

Provenzani 3, Alessandro Quattrone 3 

1 Center for Integrative Biology- University of Trento, Italy 

2 Center for Materials and Microsystems, Fondazione Bruno Kessler, Povo (Trento), Italy 

3 Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento, 

Italy 

The precise assembly of native eukaryotic polysomes and the organization of ribosomes along 

the mRNA are still obscure. Despite different imaging approaches have been used to visualize 

bacterial and eukaryotic polysomes, human native polysomes have never been observed in 

physiological conditions. Nothing is known, moreover, about formation of the 

mRNA-ribosomes macroassembly. 

Atomic Force Microscopy (AFM) is a powerful technique to image biomolecules under 

near-native conditions, and without the addition of any fixing or external contrast agent. We 

employed an innovative approach to visualize polysomes from three mammalian cell lines (rat 

PC12, human MCF7 and HeLa cells) by AFM. After isolation by sucrose gradient centrifugation 

polysomal fractions have been characterized both in liquid and air, with a protocol aimed to 

maximally preserve their original assembly. Structural parameters of the ribosomes measured 

in these conditions (height, width and area) are compatible with those previously measured by 

AFM and electron microscopy (EM). 

For the first time to our knowledge, we could be able to observe ribosome aggregates 

associated to filaments compatible with single-stranded and double stranded RNA, providing 

therefore the imaging of intact polysome assemblies. The number of ribosomes for polysome 

ranges, depending on the density of the fraction analyzed, from 1-2 to more than 25 

ribosomes. Despite rat and human ribosomes display some differences in the structural 

parameters, the overall spatial arrangement in the polysome is conserved. 

Our imaging approach questions the proposed double raw model for polysome morphology 

obtained by EM, suggesting the existence of a complex pattern of conformations. The 

systematic analysis of polysomal aggregates by this near-native methodology could reveal 

novel levels of polysomal organization, and provide structural testing of the current view about 

polysome physiology derived from biochemical data. 

264

VIVIANA VOLTA 

Just a Minute? 

Viviana Volta, Anne Beugnet, Laura Magri, Daniela Brina, Simone Gallo, Pier Carlo 

Marchisio, Stefano Biffo 

San Raffaele del Monte Tabor Foundation, Italy 

265 


RACK1 (Receptor for Activated C Kinase 1) is a scaffold protein belonging to the WD40 family. 

First characterized as a receptor for active PKCβII, RACK1 is also a structural component of 

the small ribosomal subunit, residing close to the mRNA exit channel, and to eIF3 binding site. 

In mammalian cells the RACK1/ PKCβII complex phosphorylates the initiation factor eIF6 

allowing its release from the large subunit and the formation of 80S complex. Either inhibition of 

active PKCβII binding to RACK1 or RACK1 downregulation by RNAi impairs cell response to 

translation stimulation. We decided to investigate the physiological relevance of RACK1 by 

creating a knockout mouse for RACK1. In the first attempt to flox the gene we obtained a 

mouse, which has a 200 bp deletion in addition to other alterations in the intronic regions (a 

neomycin cassette, two Frt and one LoxP sites). Mice carrying these alterations in 

heterozygosity resemble some features of Belly Spot and Tail (Bst) rpL24 mutant. Bst mutant is 

the first Minute found in mouse. Initially described in Drosophila melanogaster, Minute 

phenotype is caused by mutations in genes coding for ribosomal proteins. The mutants share 

three common features (a 2-3 day delay in larval development, short thin bristles and recessive 

lethality) and other variable phenotypes (e.g. small body size). In mouse, besides Bst mutants 

alterations in genes coding for ribosomal proteins cause decreased erythropoiesis, defects in 

pigmentation, and less frequently small size and somatic malformations. Heterozygous mice for 

RACK1 mutation exhibit a white ventral spot, close to midline and of variable size, and 

pigmentation defects on the tail. The mutation is lethal in homozygosity. No alteration in weight 

is present in adult mice. Further work is needed to better define other phenotypic features. The 

data suggest that even small alterations in the genomic region containing RACK1 gene are 

crucial



VACLAV VOPALENSKY 

Capping enzyme encoded by Kluyveromyces lactis linear plasmids doesn’t 

support cap-dependent translation of their mRNAs 

Martin Pospisek, Vaclav Vopalensky 

Charles University in Prague, Czech Republic 

Linear plasmids were found in a number of yeast species belonging to nine genera. The 

genetic organization of yeast linear plasmids appears to be quite uniform with most thoroughly 

studied plasmids pGKL1 and pGKL2 from the yeast Kluyveromyces lactis. Gene functional 

analysis has shown that pGKL1 is instrumental in the expression of a killer toxin, whereas 

pGKL2 provides vital maintenance functions for both plasmids. 

Genes encoded by pGKL plasmids are likely to be transcribed independently; mRNAs 

transcribed from the pGKL plasmids are supposed to be capped and not to be 

polyadenylated. 

Here we present a molecular analysis of a putative capping enzyme encoded by K2ORF3. We 

produced K2Orf3p as a HIS- or GST-tagged fusion protein both in E. coli and baculovirus 

expression systems, purified all the overexpressed protein variants by affinity chromatography 

to almost homogeneity and successfully tested them for their guanylyltransferase activities. 

Surprisingly, we were not able to detect any N7-methyltransferase activities of any of the 

purified K2Orf3p protein. 

We show that pGKL specific mRNAs do not bind to the cap-binding translation initiation factor 

4E from S. cerevisiae in vitro while cellular mRNAs do. This result is further supported by our 

finding that killer toxin, naturally encoded by pGKL plasmids, is translated by cap-independent 

pathway, while control killer toxin gene artificially expressed under the control of the strong Pol 

II driven promoter is translated by cap-dependent pathway. Possible variants of the 5' end 

structures naturally occurring at pGKL-specific mRNAs will be discussed. 

266

ADAM WALLACE 

Nematode Trans-Spliced Leader Sequence and Structure Required for 

Translation of TMG-Capped mRNAs 

267 


Adam Wallace 1, Megan Filbin 1, Bethany Veo 1, Richard Davis 1, Janusz Stepinksi 2, Marzena 

Jankowska-Anyszka 2, Edward Darzynkiewicz 2 

1 University of Colorado School of Medicine, United States of America 


Spliced leader RNA trans-splicing adds a small exon (the spliced leader, SL) with an atypical 

cap to pre-mRNAs to form the mature 5’ ends of recipient RNAs. In nematodes, the 22 nt SL 

cap is a trimethylguanosine cap (TMG, m2,2,7GpppG). Nematode translation of TMG-capped 

RNA is inefficient in the absence of the SL sequence compared to TMG-capped mRNAs with 

the SL. Here we define sequence and structure within the nematode SL that contributes to the 

efficient translation of TMG-capped mRNAs. The identified elements do not facilitate translation 

of m7G-capped RNAs in nematodes or TMG-capped mRNAs in heterologous translation 

systems such as rabbit reticulocyte or wheat germ. Thus, this cooperative interaction between 

a cap and the SL is specific for the TMG cap and nematode translation. Spacing of nucleotides 

within the SL and the distance of these nucleotides from the cap are important for efficient 

translation. Key sequences in the SL form a small, discrete stem loop required for translation. 

These elements are conserved in other nematode SLs (SL2-like) and correspond to regions of 

SL1 required for early C. elegans development. The SL enhances TMG cap translation at the 

level of translation initiation downstream of the cap-binding step. These studies define key 

elements within an mRNA 5’ UTR required for the efficient cap-dependent translation of 

atypical TMG-capped mRNAs.



DOMINIQUE WEIL 

Unravelling the ultrastructure of stress granules and associated P-bodies in 

human cells 

Dominique Weil, Sylvie Souquere, Stéphanie Mollet, Michel Kress, François Dautry, Gérard 

Pierron 

CNRS, France 

Stress granules (SGs) are cytoplasmic ribonucleoprotein granules formed following various 

stresses that inhibit translation (oxidative stress, heat shock, UV irradiation). They can also 

assemble following overexpression of translational regulators, such as CPEB1 and TIA1, as 

well as after virus infection. SGs contain untranslated mRNAs and are thought to help 

protecting them until stress relief. In fluorescence microscopy, SGs are frequently seen 

adjacent to P-bodies. These ribonucleoprotein foci of smaller size contain mRNAs and proteins 

involved in mRNA degradation and mRNA storage, including the RNA interference machinery. 

We have previously reported that SG assembly often takes place at the contact of pre-existing 

P-bodies. Similar observations have recently been made in yeast, raising the possibility that 

SGs and P-bodies are structurally related. We addressed this question by analysing their 

ultrastructure by electron microscopy. We show that SGs resulting from oxidative stress, 

heat-shock or protein overexpression are loosely organised, fibrillo-granular aggregates of a 

moderate electron density. By comparison, P-bodies have a fibrillar, compact structure. By in 

situ hybridization with 18S and 28S probes at the electron microscopic level, we confirm the 

presence of the small but not the large ribosomal subunit in SGs. Using a polydT probe, we 

show that SGs are enriched in polyA+ mRNA, though these represent a minor fraction of the 

cellular mRNAs. When induced by overexpression of specific translational repressors, SG 

assembly appears clearly restricted to cells in which polysomes are massively disrupted into 

monosomes. Finally, providing here the first ultrastructural characterization of SGs in eukaryotic 

cells, we show that, despite close contact with P-bodies, both domains remain structurally 

distinct and do not interdigitate. 

268

STEN WIE 

Translational Regulation of the Thymidylate Synthase mRNA 

Les Krushel, Sten Wie 

University of Colorado Denver, United States of America 

269 


Thymidylate synthase (TS) is the rate limiting step in the thymidine synthesis pathway. TS 

expression is up regulated in many cancers and its protein level correlates negatively with the 

efficaciousness of chemotherapeutic agents directed against it (e.g. 5-fluorouracil). 

Consequently, identifying the mechanism by which TS mRNA is translated may elucidate a 

novel mechanism to control TS protein levels. A major determinant of TS mRNA translation is 

thought to occur through the 5’ untranslated region (UTR). The 5’ UTR ranges from 152-180 

nucleotides in length and has 80% GC nucleotide content. Additionally, it contains two to three 

28 nucleotide contiguous repeats (TS 2R, TS 3R) and a inverted 28 nucleotide inverted repeat 

upstream. In one major allele, there is a C to G substitution with the third repeat (TS 3R C→G). 

We assayed the translatability of the TS 5’ UTRs by placing them upstream of the Photinus 

luciferase ORF and transfecting capped and tailed mRNA into HeLa cells. RNA levels were 

quantified by QRT-PCR and protein expression by Photinus luciferase activity. The mRNA with 

the TS 3R (C→G) 5’ UTR generated the highest level of protein synthesis followed by TS 3R 

and TS 2R. In addition, the level of total protein synthesis from the three mRNAs was roughly 

equivalent to a mRNA with a short 5’ UTR. The inclusion of G/C nucleotide repeats would not 

be predicted to enhance translation initiation via the cap-scanning model. Indeed our 

preliminary data using dicistronic and ApppG capped RNAs indicate that the TS 5’ UTR 

contains an IRES. Our present goal is to verify the presence of an IRES and determine if the 

secondary structure of the 5’ UTR is necessary for IRES activity.



HANS-JOACHIM WIEDEN 

Emerging roles of the universally conserved GTPases HflX and YchF during 

protein synthesis 

Hans-Joachim Wieden, Jeffrey Fischer, Alvin Altamirano, Michael Shields 

University of Lethbridge, Canada 

The bacterial P-loop GTPases YchF and HflX are the only members of a group of 8 universally 

conserved GTPases whose function are still unknown. Most members of this group of 

GTPases are involved in protein synthesis (e.g. EF-G/EF2, EF-Tu/EF-1α, IF2, Ffh and FtsY). 

YchF has been shown to be essential for a number of organisms like S. pneumoniae and S. 

aureus. However it’s cellular function remains to be elucidated. HflX has previously been shown 

to associate with the 50S ribosomal subunit and both proteins bind and hydrolyse ATP. Here 

we provide for the first time evidence towards understanding the cellular function of these two 

proteins. YchF forms a stable complex with the large subunit ribosomal RNA in-vivo. This 

complex can be reconstituted in-vitro with nM affinity. Most interestingly, we not only confirm 

that E. coli YchF hydrolyses ATP more efficient than GTP, but also that binding of the large 

subunit ribosomal RNA to YchF is able to stimulate its ATPase activity significantly. In an effort 

to elucidate the cellular function of HflX, we have determined the kinetic parameters governing 

the interaction between HflX and GTP/ATP nucleotides using fluorescence based equilibrium 

and pre-steady state techniques. Based on the obtained kinetic parameters, we demonstrate 

that the GTPase activity of HflX is stimulated by 50S ribosomal subunits, as well as by empty 

and poly(U) programmed 70S ribosomes. Interestingly, the 70S stimulated GTPase activity is 

specifically inhibited by the antibiotic chloramphenicol, which binds to the large ribosomal 

subunit, but not by kanamycin, an aminoglycoside targeting the small ribosomal subunit. 

270

JIM WILHELM 

271 


The eIF4E binding portein Hubcap defines a novel class of localized RNPs 

Jim Wilhelm, Elena Monfort-Prieto, Risa Maruyama, Brian Sato 

UC San Diego, United States of America 

Translational control of localized messenger RNAs (mRNAs) is critical for cell polarity, synaptic 

plasticity, and embryonic patterning. In Drosophila oogenesis, a core complex comprised of 

the RNA helicase, Me31B, the eIF4E binding protein, Cup, the Y box RNA binding protein, 

Yps, and the Lsm family RNA binding protein, Trailer Hitch (Tral) is believed to translationally 

repress maternal messages. Consistent with this complex playing a role in the translational 

control of localized messages, cup and me31b are required for translational repression of the 

posteriorly localized message oskar (osk). However, the tral subunit is not required for 

regulation of osk mRNA and, in fact, has defects in the secretion of the EGF receptor ligand, 

Grk – a key regulator of dorsal-ventral patterning. The radically different phenotypes 

associated with components of the same complex suggested that there might be a second 

Tral-containing complex that could explain the phenotypes observed in tral mutants. In order 

to identify additional Tral complexes, we used a proteomics approach to identify all of the Tral 

associated proteins in the early embryo. This approach identified a previously uncharacterized 

protein, Hubcap, a novel eIF4E binding protein that is localized within the oocyte. Furthermore, 

dominant-negative forms of hubcap disrupt dorsal-ventral patterning suggesting that 

alterations in the Hubcap complex may underlie the tral phenotype. Consistent with this 

interpretation, the localization of Hubcap complexes is completely dependent on tral, while the 

localization of other complexes is not. Biochemical studies of Hubcap complexes isolated 

from tral mutant ovaries revealed that the localization defect is not due to a failure to assemble 

the Hubcap RNP. Thus, Hubcap defines a novel class of translational control complexes 

whose localization is tral-dependent.



MARK WILLETT 

Translational Control during Cell Spreading, Adhesion and Migration 

Mark Willett, Rachel Barrett, Markete Vlasak, Hilary Pollard, Jenny Pain, Simon Morley 

University of Sussex, United Kingdom 

The migration of eukaryotic cells involves the modulation of complex regulatory signaling 

pathways which mediate the spatial and temporal overall polarisation of the cell, cytoskeletal 

organisation, and the formation and turnover of cell-matrix and cell-cell adhesion receptors. 

This process contributes to the regeneration and repair of tissues, as well as orchestrating 

embryonic morphogenesis. Mis-regulation of cell migration can result in disease progression, 

such as increased metastasis and invasiveness in cancer, mental retardation due to impaired 

embryonic development, atherosclerosis and arthritis. 

Although much effort has been made to elucidate the cellular systems that contribute to this 

process, little is known as to the role of translation. Our work seeks to elucidate the 

mechanisms of transport and compartmentalisation of the translational machinery, as well as 

the function of localised translation in cell adhesion, polarity and migration. 

This work was funded by the BBSRC 

272

ANNE WILLIS 

273 


Polypyrimidine tract binding protein is a regulator of cytoskeletal organisation 

and cell migration 

Gant Tim 1, Laura Cobbold 2, Anne Willis 2, Kirsty Sawicka 2, Keith Spriggs 2, Bushell Martin 2, 

Ruth Spriggs 2 

1 MRC-Leicester, United Kingdom 


Polypyrimidine tract binding protein (PTB) is a multifunctional RNA binding protein that shuttles 

between the nucleus and the cytoplasm. In the nucleus it functions as a splicing factor, 

whereas in the cytoplasm it has roles in mRNA stability, localisation and translation. To define 

the full complement of mRNAs with which PTB interacts and whose translation is affected by 

PTB we have carried out polysome profiling following knock down by RNAi of PTB, nPTB, and 

nPTB combined with PTB, in conjunction with RNA-immuno-precipitation using a PTB specific 

antibody. Interestingly, approximately 25% of the mRNAs identified by these screens (the 

largest functional group) have a role in cytoskeletal organisation and cell migration including 

BRAF, RhoA, Vimentin, Profilin and ARPC2. We have shown that PTB directly regulates the 

expression of these proteins since knockdown of PTB and nPTB reduces both the polysomal 

association and the translational efficiency of these mRNAs. The data also show that PTB is 

required for localisation of some of these mRNAs. Thus following serum stimulation RhoA, 

Vimentin and Profilin mRNAs relocalise to the leading edge of the cell and reduction in PTB 

expression by siRNA inhibits this process. Consistent with a role for PTB in cytoskeletal 

organisation/cell migration, inhibition of the localised translation of these mRNAs by PTB 

knockdown has a dramatic effect on cell shape and there is a complete inhibition of cell 

migration under these conditions. These data show that cytoplasmic PTB is able to 

post-transcriptionally regulate expression of a defined subset of mRNAs and provide an 

explanation for the association of increased expression of this protein in metastatic tumours.



ANNIKA WOLF 

Plakophilin 1 stimulates cell proliferation and growth by promoting eIF4A 

activity 

Annika Wolf, Mechthild Hatzfeld, Malgorzata Krause-Gruszczynska 


Plakophilins 1-3 are desmosomal proteins of the p120ctn-family of armadillo related proteins 

that are essential for organising the desmosomal plaque. Recent findings identified plakophilins 

in stress granules suggesting an association with RNA-binding proteins and components of the 

translational machinery. However, a function of plakophilins in controlling translation remained 

elusive. In an attempt to characterize the potential role of plakophilin 1 in this process, we 

identified the eukaryotic translation initiation factor (eIF) 4A1 as a putative interaction partner in 

a yeast two-hybrid screen. Plakophilin 1 associated directly with eIF4A1 in vivo and in vitro. 

During cellular stress, both proteins were recruited to stress granules. In non-stressed cells, 

endogenous plakophilin 1 coprecipitated together with eIF4A1 and eIF4E in the cap-binding 

initiation complex. The overexpression of plakophilin 1 led to increased overall translation rates, 

whereas plakophilin 1 knockdown had the opposite effect. The stimulation of translation upon 

plakophilin 1 overexpression correlated with its capacity to promote eIF4A1-ATPase-activity in 

vitro. Moreover, plakophilin 1 stimulated the recruitment of eIF4A1 into translation initiation 

complexes in vivo. The effects of plakophilin 1 overexpression on translation correlated with the 

upregulation of cell proliferation and cell size. These findings identify plakophilin 1 as a regulator 

of translation initiation and suggest that plakophilin 1 promotes cell proliferation by stimulating 

protein synthesis through its interaction with eIF4A1. 

274

CONNIE WAI HONG WOO 

275 


Prolonged Physiologic ER Stress Triggers Adaptive Suppression of 

ATF4-CHOP by a Mechanism that Appears to "Compensate" for the Protein 

Translational Effects of P-eIF2 alpha 

Connie Wai Hong Woo, Ira Tabas 


The Unfolded Protein Response (UPR) restores protein folding equilibrium to the endoplasmic 

reticulum (ER), in part by inducing eIF2a phosphorylation, which inhibits eIF2B and limits 

ternary complex (TC). Decreased TC helps to restore ER equilibrium by temporarily 

suppressing global translation while inducing translation of the UPR effector ATF4. Despite the 

adaptive nature of this pathway in short-term ER stress, prolonged expression of the ATF4 

target CHOP is cytotoxic. Thus, under prolonged physiologic ER stress, we hypothesized that 

cells must somehow selectively suppress the ATF4-CHOP pathway. Using innate immunity as 

a prolonged UPR model, we found that TLR signaling suppresses ATF4-CHOP in cells 

subjected to prolonged ER stress, but PERK and eIF2a phosphorylation (upstream of ATF4) 

were not suppressed. The translation of ATF4 was suppressed, despite the presence of 

P-eIF2a, and global translation remained active. This unique pathway to prevent cytotoxic 

ATF4-CHOP during prolonged ER stress appears to involve a mechanism whereby cells 

"ignore" the P-eIF2a effect on translation. We hypothesized that some events in 40S ribosome 

assembly might compensate for limiting TC. We found that TLR signaling induces eIF4B 

phosphorylation. Because P-eIF4B promotes association of the eIF4F subunit with eIF3 of 

pre-initiation complex to form the 40S ribosomal subunit, this finding might explain how ATF4 

is suppressed and global translation is left intact despite P-eIF2a and limiting TC. In summary, 

cells prevent cytotoxic CHOP expression during prolonged physiologic ER stress during TLR 

signaling by suppression of ATF4 translation. The mechanism may involve "compensation" for 

the presence of P-eIF2a. This pathway may have evolved to protect cells from prolonged ER 

stress during the innate immune response.



ANNA WYPIJEWSKA 

Substrate specificity of C. elegans scavenger decapping enzyme DcpS for 

m7GpppG, m2,2,7GpppG and chemically modified dinucleotide cap analogues 

Richard Davis 1, Anna Wypijewska 2, Elzbieta Bojarska 2, Janusz Stepinski 2, Jacek Jemielity 2, 

Edward Darzynkiewicz 2, Marzena Jankowska-Anyszka 3 

1 Department of Biochemistry and Molecular Genetics, University of Colorado Health 

Sciences Center, Aurora, CO 80045, United States of America 

2 Department of Biophysics, Institute of Experimental Physics, University of Warsaw, 02-089 

Warsaw, Poland 

3 Department of Chemistry, University of Warsaw, 02-093 Warsaw, Poland 

The 7-methylguanosine cap at the 5’ end of eukaryotic mRNA plays an important role in gene 

expression. It is required for RNA translation, splicing, transport and stability. Hydrolysis of the 

free cap catalyzed by the Scavenger Decapping Enzyme (DcpS) is a critical mechanism for 

regulation of mRNA turnover. The object of our studies was to examine the activity of C. 

elegans DcpS on its natural substrates m7GpppG and m32,2,7GpppG, and dinucleotide cap 

analogues, chemically modified in the nucleoside’s base (benz7GpppG, et7GpppG, 

m7Gpppm7G, m7GpppA, m7Gpppm6A, m32,2,7GpppA) or ribose moiety (m27,2’OGpppG, 

m27,3’OGpppG, m7Gppp2’dG, m7Gpppm2’OG). The kinetic parameters of enzymatic 

hydrolysis (Km, Vmax, kcat) of these substrates catalyzed by C. elegans DcpS were 

determined using fluorescence studies and HPLC analysis. Nematode DcpS from C. elegans 

readily hydrolyzes both monomethylguanosine (m7G) and trimethylguanosine (m32,2,7G) 

containing cap structures with low micromolar Km. Cap analogues with selective modifications 

within the first transcribed nucleotide were hydrolyzed with similar kinetics as the native 

substrates, regardless of the type of purine base. Kinetic data for dinucleotides containing 

unmodified guanine and modifications to the 7-methylguanosine moiety demonstrated a crucial 

role of 2’-OH and 3’-OH groups of m7Guo for DcpS cap recognition and hydrolysis. 

benz7GpppG and et7GpppG were hydrolyzed with similar efficiency as native caps, whereas 

ARCA-type cap analogues (m27,2’OGpppG and m27,3’OGpppG) were less efficiently 

hydrolyzed by C. elegans DcpS. These new studies provide an insight into the future 

development of ARCA technology and cap analogues for biochemical studies of anti-parasite 

drugs based on the atypical m32,2,7GpppG cap in parasitic nematodes. 

276

AKIKO YANAGIYA 

277 


Characterization of the role of PABP interacting protein 2 in the late stage of 

spermatogenesis in PAIP2 knockout mouse model 

Akiko Yanagiya, Geraldine Delbes, Bernard Robaire, Nahum Sonenberg 


A number of mRNAs in the late stage of spermatogenesis are regulated under translational 

control. For example, protamines and transition proteins mRNAs are stored as translationally 

inactive messenger ribonucleoprotein particles (mRNPs) in the early haploid cells, and their 

translation are activated by shortening their mRNA poly(A) tail in late spermatogenesis. Poly(A) 

tails are associated with poly(A) binding proteins (PABPs) that are involved in multiple functions 

such as mRNA biogenesis, mRNA stabilization and translation. PABP activity is modulated by 

PABP-interacting proteins (Paips); Paip1 and Paip2. Paip2 was initially identified as a 

translational inhibitor, since Paip2 promotes the dissociation of PABP from poly(A) tail and 

competes with eIF4G for binding to PABP. This results in impairment of the cap and poly(A) tail 

causing a synergistic enhancement on translation. There are 2 homologs of Paip2, Paip2A and 

Paip2B. Both are expressed in the testis, but Paip2A is the predominant form. To investigate 

the physiological role of Paip2, single Paip2A and Paip2B knockout (KO) mice and double KO 

(DKO) mice have been generated using Cre/loxP system. We demonstrate that male Paip2A 

KO and DKO mice are infertile with almost no sperm found in the epididymis. Furthermore, we 

have shown that Paip2A is expressed only in the late haploid spermatogenic cells in the testis 

and that the elongated spermatids are not released into the lumen at the end of 

spermatogenesis when Paip2A is absent. Moreover, PABP is still expressed in the very late 

stages of spermatogenesis in the DKO mice when it is absent in wild-type. Electron 

microscopic analysis revealed an impairment of mitochondria alignment around the flagellum 

as well as chromatin condensation abnormalities in the DKO mouse. Taken together, these 

data suggest that Paip2A has an important role in translational control in the late stage of 

spermatogenesis by regulating PABP function.



EMILIO YÁNGÜEZ 

Influenza virus requirements for eIF4F components: hijacking cellular 

translation machinery 

Alfredo Castelló 1, Emilio Yángüez 2, Amelia Nieto 2, Ian Goodfellow 3 

1 Centro de Biología Molecular Severo Ochoa (CSIC), Spain 

2 Centro Nacional de Biotecnología (CSIC), Spain 

3 Imperial College London, United Kingdom 

Influenza virus mRNAs are structurally indistinguishable from cellular ones as they bear 5’-cap 

structures and are polyadenylated at their 3’-ends. However, selective translation of viral 

mRNAs, despite inhibition of host-cell protein synthesis, occurs upon infection. Initiation of 

translation is a major target for gene expression regulation and viruses have evolved numerous 

unconventional mechanisms to recruit cellular translational machinery. Often, interactions of 

viral proteins with the components of eIF4F complex and with viral mRNAs allow selective viral 

protein translation interfering host cell protein synthesis. 

Influenza virus polymerase is able to bind to cap structures and to the 5’-UTR of viral mRNAs, 

and interacts with translation initiation complexes. Here, we characterize in depth viral 

polymerase interaction with translation initiation complexes and we evaluate the influenza virus 

requirements for eIF4F translation initiation complex components. We provide evidence that 

both the viral polymerase and the isolated PB2 polymerase subunit, which possesses 

cap-binding activity, interact with translation initiation complexes. This interaction is 

independent on the presence of viral mRNA. Moreover, in vitro viral mRNA translation is strictly 

dependent on cellular cap-binding protein eIF4E while infection proceeds efficiently upon 

impairment of eIF4E function suggesting that there are no viral mRNA sequences responsible 

for this eIF4E low requirement. However, viral translation seems to be strictly dependent on 

eIF4AI and eIF4GI activities both in in vitro and in vivo studies. These results suggest the 

implication of viral polymerase in the selective translation of viral mRNA in infected cells and a 

possible independence of cellular cap-binding protein eIF4E in this process. 

278

LAURE YATIME 

Structure of the RACK1 dimer from Saccharomyces cerevisiae 

Jakob Nilsson 1, Laure Yatime 2, Kim L. Hein 2, J. Preben Morth 2, Poul Nissen 2 

1 BRIC - Copenhagen University, Denmark 

2 MBI - Aarhus University, Denmark 

279 


The Receptor for Activated C-Kinase (RACK1) serves as a scaffolding protein in numerous 

signaling complexes involving kinases and membrane-bound receptors from different cellular 

compartments. It exists simultaneously as a cytosolic free form and as a ribosomal-bound 

protein. As part of the 40S ribosomal subunit, it triggers translational regulation by establishing 

a direct link between PKC and the protein synthesis machinery. It has been suggested that 

RACK1 could recruit other signaling molecules onto the ribosome, providing a 

signaling-specific modulation of the translational process. RACK1 is able to dimerise both in 

vitro and in vivo. This homodimer formation has been observed in several processes including 

the regulation of the NMDA receptor by the Fyn kinase in the brain and the 

oxygen-independent degradation of the hypoxia-inducible factor 1. The functional relevance of 

this dimerisation is however still unclear and the question of a possible dimerisation of the 

ribosome-bound protein is still pending. Here we report the first crystal structure of a RACK1 

homodimer from Saccharomyces cerevisiae at 2.8 Å resolution. The structure reveals an 

atypical mode of dimerisation where both monomers intertwine two beta-strands from blade 4 

to form a mutually shared blade, consequently exposing a novel surface of the protein to new 

potential interacting partners. The significance of the physiological requirement for a RACK1 

dimer will be discussed.



LUCY YOUNG 

Translational reprogramming following DNA damage 

Anne Willis, Lucy Young, Alexander Kondrashov, Martin Bushell 


It has been shown previously that post-transcriptional regulation of gene expression forms an 

essential part of the DNA damage response to UV light [1]. To determine whether a similar 

mechanism is used following exposure of cells to alkylating agents HeLa cells were treated 

with a non-lethal dose of the chemical ethylating agent, ethylmethane sulfonate (EMS). 

Following exposure to EMS there was a reduction in the rate of global protein synthesis to 40% 

of that observed in control cells. The global protein synthesis inhibition was mediated by a 

phosphorylation of the eukaryotic initiation factor (eIF) 2 on the α subunit, therefore a reduction 

of ternary complex formation. To identify mRNAs subject to differential translational regulation 

following EMS exposure we used polysome profiling, a technique in which the 

sub-polysome/polysome distribution of a large number of mRNAs is analysed using cDNA 

microarray. Our microarray data suggests there is an upregulation of mRNAs encoding genes 

involved in cellular stress responses including ATF4 and Metallothionein; cytoskeletal 

organisation (α-tubulin and Thymosin); regulation of translation (PABP and ZFP36L1); DNA 

repair and cell cycle control (SHFM1 and CDKN2C). These data have been confirmed by 

northern analysis which shows these mRNA to be associated with an increased number of 

ribosomes and western analysis which demonstrates a complementary increase in protein 

levels. Interestingly, we have observed a delay in cell cycle progression via a G2/M arrest 

following EMS exposure, which may correlate to the observed up-regulation of specific 

cyclin-dependent kinase inhibitors. Sequence elements within the 5’ and 3’UTRs of these 

mRNAs have been identified that could contribute to the translational regulation of the 

endogenous mRNAs following EMS exposure and experiments are currently being performed 

to determined their role in this process. 

[1] Powley, I., et al. (2009) Genes Dev. 23(10):1207-1220 

280

IZABELA ZABOROWSKA 

281 


A role for Vaccinia Virus I3L in the redistribution of host translation factors to 

cytosolic viral replication compartments 

National Institute for Cellular Biotechnology, Ireland 

Poxviruses are large, double-stranded DNA viruses that replicate exclusively in the cytosol. 

Recent reports have shown that Vaccinia Virus (VacV), a prototypical poxvirus, recruits eIF4E 

and eIF4G into cytoplasmic viral factories that become surrounded by PABP. These events are 

sensitive to phosphonoacetic acid (PAA), an inhibitor of the viral DNA polymerase, suggesting 

that a late viral gene product or function linked to viral DNA replication mediates translation 

factor relocalisation. Using immunoprecipitation and mass spectrometry to identify translation 

factor-associated proteins in infected cells we have identified I3L, a VacV-encoded 

ssDNA-binding protein, as specifically associating with eIF4G but not PABP, two translation 

factors that differentially localize during infection. GFP-tagged I3L expressed in 293 cells shows 

diffuse cytoplasmic staining that rapidly relocalises upon infection to discrete punctate 

structures in viral factories that co-localise with eIF4G, a process that is sensitive to PAA. 

VacV-encoded or FLAG-tagged I3L associate with cap-bound eIF4F complexes in either 

infected or uninfected, I3L-expressing 293 cells, respectively. In uninfected 293 cell extracts 

FLAG-tagged I3L has the capacity to recruit translation factors to exogenously-added ssDNA. 

Finally, I3L expression in VacV-infected cells in insensitive to treatment with PAA. Our results 

present a temporal image of events whereby production of a virus-encoded ssDNA binding 

protein and its’ association with specific host translation factors precedes the formation of 

cytoplasmic viral replication compartments, where viral DNA and RNA synthesis occurs. We 

propose that this enables rapid and selective movement of factors to viral factories upon their 

formation and suggest that the effect of PAA on translation factor redistribution is due to the 

inhibition of ssDNA production within these sites, which would act as a localisation signal for 

I3L-bound translation complexes.



YONGLONG ZHANG 

Characterization of YafO, an Escherichia coli toxin 

Yonglong Zhang, Yoshihiro Yamaguchi, Masayori Inouye 

UMDNJ-Robert Wood Johnson Medical School, United States of America 

YafO is a toxin encoded by the yafN-yafO antitoxin-toxin operon in the Escherichia coli 

genome. Our results show that YafO inhibits protein synthesis, but not DNA and RNA 

synthesis. The in vivo [35S]methionine incorporation was inhibited within 5 min after YafO 

induction. In in vivo primer extension experiments with two different mRNAs, the specific 

cleavage bands appeared 11-13 bases downstream of the initiation codon, AUG, at 2.5 min 

after the induction of YafO. An identical band was also detected in in vitro toeprinting 

experiments when YafO was added to the reaction mixture containing 70S ribosomes and 

same mRNAs even in the absence of tRNAfMet. Notably, this band was not detected in the 

presence of YafO alone, indicating that YafO by itself does not have endoribonuclease activity 

under the conditions used. The full-length mRNAs almost completely disappeared at 30 min 

after YafO induction in in vivo primer extension experiments, consistent with Northern blotting 

analysis. Over 84% of [35S]methionine-tRNAfMet was released from translation initiation 

complex at 5.43 mM YafO in vitro. We demonstrated that the 70S ribosome peak significantly 

increased upon YafO induction, and when the 70S ribosomes dissociated into 50S and 30S 

subunits, YafO was found to be associated with 50S subunits. These results demonstrate that 

YafO is a ribosome-dependent mRNA interferase primarily inhibiting protein synthesis. 

282

SONJA ZILOW 

Upstream of N-Ras (Unr) is involved in Translational Control of ADAM10 

283 


Elisabeth Kremmer 1, Dominik Buell 2, Ann-Katrin Ludwig 2, Ignasi Forne 2, Axel Imhof 2, 

Christian Haass 2, Sven Lammich 2, Sonja Zilow 2, Helene Jacquemin-Sablon 3 

1 Helmholtz Zentrum, Munich, Germany 

2 Ludwig-Maximilians University, Munich, Germany 

3 Victor Segalen University, Bordeaux, Germany 

Alzheimer disease is characterized by the accumulation of amyloid plaques and neurofibrillary 

tangles in the brain. Plaques mainly consist of the 40 or 42 amino acid amyloid-beta peptide 

(Abeta) which is derived via proteolysis of the amyloid precursor protein (APP) by beta- and 

gamma-secretase. In contrast alpha-secretase prevents the formation of Abeta by cleaving 

APP within the Abeta domain. There is strong evidence that ADAM10 (a disintegrin and 

metalloproteinase) acts as alpha-secretase in vivo. ADAM10 has a 444 nucleotides conserved 

5ùntranslated region (UTR) with potential upstream open reading frames and a GC content of 

70%. We demonstrate that the 5’UTR of ADAM10 represses ADAM10 translation. Thus 

ADAM10 expression may be regulated at the posttranscriptional level via 5’UTR binding 

proteins. To identify possible binding candidates we performed a UTR-database search and 

affinity chromatography. Using these two approaches, we identified Unr, a cytosolic RNA 

binding protein. We provide evidence that Unr binds to the 5’UTR of ADAM10 using 

electrophoretic mobility shift assays. Deletion of a 83 purine-rich nucleotide stretch within the 

5’UTR abolished the binding of recombinant Unr to the 5’UTR. of ADAM10. Moreover upon 

Unr knockdown reduced levels of ADAM10 as well as reduced ADAM10 mRNA are observed. 

Due to the reduced ADAM10 protein levels, the ADAM10 dependent shedding of APP is 

lowered. In this study we demonstrate that Unr binds to the 5’UTR of ADAM10 and that Unr is 

involved in translational regulation of ADAM10 protein expression.



JOANNA ZUBEREK 

Binding specificities of multiple Drosophila eIF4E family members to cap 

structure 

Nahum Sonenberg 1, Greco Hernandez 2, Jacek Jemielity 3, Edward Darzynkiewicz 3, Joanna 

Zuberek 4 

1 Department of Biochemistry and McGill Cancer Center, McGill University, Canada 

2 Department of Biology and Department of Biochemistry, McGill University, Canada 

3 Division of Biophysics, Institute of Experimental Physics, University of Warsaw, Poland 


Translational control is critical for proper regulation of cell cycle, growth, embryogenesis and 

germ-line development. The control is achieved through variety actions where initiation is an 

important event. The cap-dependent translation begins with eukaryotic initiation factor (eIF) 4E 

recognizing the 7-methylG(5’)ppp(5’)N cap structure at the 5’ terminus of the mRNA. Most 

eukaryotic organisms express multiple eIF4E family members classified in three main structural 

classes. Some of the isoforms such as canonical eIF4E (currently termed eIF4E-1 or eF4E-1a) 

are involved in translation initiation in general while other in repression of translation of specific 

mRNAs. Despite a number of recent advances the physiological role of most isoforms remains 

to be discovered. 

In Drosophila, there are seven genes encoding eight eIF4E proteins. Seven of them belong to 

class I, and one 4EHP (eIF4E-8) to class II. In our studies, fluorescence titration method was 

employed to characterize the binding affinity of deIF4E family members for several cap analogs. 

The ability to bind m7GTP by two isoforms: eIF4E-1 and eIF4E-2 (products of alternative 

splicing of the primary transcript) is comparable to each other, however about 8-fold weaker 

than to human eIF4E-1. Unexpectedly, it has been found that the d4EHP, repressor of caudal 

and hunchback mRNA translation, binds m7GTP only 3-fold weaker than deIF4E-1, whereas in 

the case of their human counterparts this difference is 100-fold. 

These results suggest that in Drosophila other mechanisms (e.g. low protein level) rather than 

the low cap binding affinity of d4EHP prevent from competing with deIF4E-1 for association 

with the mRNA cap to inhibit global translation. 

284

A 

285 

Authors Index 

Abaza, Irina 40 

Abraham, Christopher 213 

Achsel, Tilmann 68 

Adam, Pont 48 

Adeli, khosrow 152 

Aebersold, Ruedi 149 

Afonina, Zhanna 236 

Agirrezabala, Xabier 62 

Ainaoui, Nadera 27 

Aït-Abdellah, Samira 185 

Akbergenov, Rashid 21, 69 

Albert, Weixlbaumer 64 

Alekhina, Olga 236 

Al-Jubran, Khalid 81 

Alkalaeva, Elena 160, 236 

Allain, Frédéric 118, 181 

Allamand, Valérie 167 

Allison, Rachel 222 

Allmang, Christine 165 

Altamirano, Alvin 270 

Altman, Roger 17, 102 

Amaral, Margarida D 219 

Amthor, Beate 54 

Anderson, Ross 70 

Andreev, Dmitry 71 

Antih, Nicolas 110 

Antoniazi, Simone A 254 

Anuradha, Seshadri 260 

Araud, Tanguy 98


EMBL Heidelberg, 09– 13 2009 

Arif, Abul 126 

Arju, Rezina 109 

Armache, Jean-Paul 19 

Armisen, Javier 26 

Arribere, Joshua 72 

Arseni, Natalia 264 

Artus-Revel, Caroline 22 

Ascano, Manuel 58 

Atkins, John 93 

Atkinson, Gemma C. 138 

Atzmon, Andrea 217 

Avdulov, Svetlana 46 

Azar, Rania 27 

B 

Bogdanov, Alexey 132 

Badura, Michelle 73 

Bag, Jnanankur 78 

Bagni, Claudia 107 

Balagopal, Vidya 74 

Baldi, Odette 134 

Ban, Nenad 67, 79 

Barbosa, João A. R. G. 228 

Barends, Sharief 8 

Barna, Maria 32 

Barreto, Celeste 219 

Barrett, Rachel 272 

Bartel, David 2, 136 

Barthet-Barateig, Adeline 106 

Bartoli, Kristen 20 

Bastide, Amandine 75 

286

287 

Authors Index 

Baumann, Sebastian 34 

Baus, Diane 28 

Beattie, Kenneth A 254 

Becker, Annemarie 158 

Becker, Thomas 19 

Beckmann, Roland 19 

Begay, Valerie 169 

Beilharz, Traude 24 

Belew, Ashton 55 

Belsham, Graham 76 

Ben-Dror, Iris 217 

Bennett, Keiryn 50 

Bennink, Jack R. 103 

Benyumov, Alexey 77 

Bertea, M. 69 

Beugnet, Anne 265 

Bhattacharya, Rumpa 78 

Bhattacharyya, Suvendra 22 

Bhushan, Shashi 18 

Biffo, Stefano 134, 224, 265 

Bingel-Erlenmeyer, Rouven 67 

Binninger, Petra 87 

Biondini, Laura 176 

Bitterman, Peter 46, 77 

Blackshear, Perry 144 

Blanchard, Scott 17, 102 

Blanzieri, Enrico 218 

Blenkiron, Cherie 149 

Bochkaeva, Zanda 203 

Boehringer, Daniel 67, 79



Boel, Gregory 43 

Bogan, Erica 149 

Bogdan, Christian 177 

Bojarska, Elzbieta 276 

Bono, Fulvia 53 

Boor, Ilja 231 

Böttger, Erik 21, 69 

Bottley, Andrew 80 

Boulo, Thomas 196 

Bour, Tania 127 

Boye, Erik 155 

Bradrick, Shelton 41 

Brandi, Anna 11 

Braun, Jörg 57, 137 

Braunegger, Nico 49 

Breitenbach-Koller, Lore 55 

Bremer, Anna 86 

Brierley, Ian 173, 206, 214 

Brill, Laurence M. 12 

Brina, Daniela 134, 265 

Brogna, Saverio 81 

Brook, Matthew 70, 82, 133 

Brown, Jeremy 115 

Brown, T. David K. 214 

Brünger, Katharina 96 

Bryant, Helen 13 

Buckingham, Richard 139 

Buell, Dominik 283 

Bueno-Martínez, José 135 

Bugiani, Marianna 231 

288

289 

Authors Index 

Bukau, Bernd 226 

Büll, Dominik 163 

Bullock, Simon 35, 168 

Bung, Christiane 203 

Burger, Lukas 58 

Burgess, Hannah 133 

Bushell, Martin 75, 95, 280 

Byström, Anders 84 

C 

Cabrera, Rodrigo 12 

Cajigas, Iván 85 

Caldarola, Sara 176 

Calkhoven, Cornelis 86, 169, 193 

Callahan, Robert 47 

Camacho-Villasana, Yolanda 208 

Cannell, Ian 83 

Cao, Yu 28 

Carmi, Asaf 258 

Carmo-Fonseca, Maria 110 

Carotti, Marcello 61 

Carriço, Renata 212 

Carrier, Marilyn 88 

Carvalho, Ana Luisa 221 

Carvalho, Joana 212 

Casanova, Claudia 87 

Castelló, Alfredo 278 

Castilho, Beatriz A. 228 

Catallo, Régine 91 

Cate, Jamie 17, 59, 117 

Cencic, Regina 88



Chang, Eric C. 12 

Chaudhury, Arindam 141 

Chavatte, Laurent 89 

Chen, Changchun 90 

Cheung, Yuen_Nei 199 

Chien, Wei Wen 91 

Chiluiza, David 47 

Chipot, Christophe 255 

Chirkova, Anna 92 

Chung, Betty 93 

Cimarelli, Andrea 241 

Clancy, Jennifer 24 

Clarkson, Bryan 94 

Claudia, Baierlein 159 

Clemens, Michael 106 

Clementi, Nina 92 

Closs, Ellen I. 22 

Cloutier, Serge 254 

Cobbold, Laura 273 

Coll, Olga 37 

Collinson, Ian 67 

Combredet, Chantal 156 

Conte, Caroline 27 

Conti, Elena 51, 53 

Cook, Amy 33 

Cooperman, Barry S. 151 

Coordes, Britta 96 

Costa, Maria 225 

Cottevielle, Magali 43 

Crépieux, Pascale 196 

290

291 

Authors Index 

Cruz, Angela K 254 

Cuchalova, Lucie 97 

Curran, Joseph 98 

Czech, Andreas 99 

D 

Dontsova, Olga 132 

Dakshinamurthy, Arun 45 

D'Antonio, Maurizio 195 

Danyi, Istvan 194 

Darvishian, Farbod 109 

Darzynkiewicz, Edward 42, 146, 157, 161, 162, 174, 202, 246, 251, 267, 276, 284 

Darzynkiewicz, Zbigniew 146, 157 

Das, Suman 103 

Daugeron, Marie-Claire 101 

Dautry, François 268 

Dave, Richa 102 

David, Alexandre 103 

David, Tscharke 248 

Davis, Cole 225 

Davis, Richard 174, 267, 276 

de Colibus, Luigi 104, 243 

de Melo Neto, Osvaldo 105 

de Moor, Cornelia 106 

De Rubeis, Silvia 107 

de Vries, Sebastian 108 

Deborah, Silvera 109 

Debus, Andrea 177 

Decimo, Didier 222, 241 

Defina, Tânia PA 254 

Delbes, Geraldine 277



Delluc-Clavieres, Aurelie 27 

Demeshkina, Natalia 63 

Demina, Irina 132 

Dessaigne, Sabine 185 

Desterro, Joana 110 

Dever, Thomas 111 

Dewell, Scott 58 

Dey, Madhusudan 111 

Dikstein, Rivka 9 

Dimitrova, Lyudmila 143 

Ding, Xavier 149 

Dinman, Jonathan 55 

Dmitriev, Sergey 71, 250 

Dobrikova, Elena 112 

Dobson, Tara 113 

Doller, Anke 114 

Dori, Landry 253 

Doronina, Victoria 115 

Dossin, Fernando M 254 

Doudna, Jennifer 94, 147 

Dougherty, Jonathan 116 

Driscoll, Donna 89 

Dubbaka, S.R. 21 

Dueggeli, Regula 22 

Duffin, Ruth 106 

Duncan, Kent 39 

Dunkle, Jack 59 

Durand, Guillaume 196 

Duss, Olivier 118 

Dziembowski, Andrzej 246 

292

E 

293 

Authors Index 

Eberhardt, Wolfgang 114 

Eberle, Andrea 52 

Ebert, Judith 53 

Eichwald, Sabrina 193 

Elantak, Latifa 261 

Eldad, Naama 119 

Elisa, Izaurralde 137 

Eliseeva, Irina 120 

Endo, Kei 121 

Eriani, Gilbert 8 

Erlacher, Matthias 92 

Esberg, Anders 84 

Eulalio, Ana 23 

Everson, Richard 131 

Fabian, Marc 147 

Fahrenholz, Falk 163 

Fan, Danhau 46 

Farabaugh, Philip 180 

Farinha, Carlos 219 

Fawcett, J 29 

Fedyunin, Ivan 122 

Feldbrügge, Michael 34 

Feltri, M. Laura 195 

Fernandez, D. 21 

Ferreira, Tiago R 254 

Ferreiro, Ana 167 

Fetcher, Pierre 60 

F



Ficner, Ralf 154 

Figaro, Sabine 139 

Figueiredo, Regina C. B. Q. 105 

Filbin, Megan 267 

Filipowicz, Witold 22, 110 

Firczuk, Helena 13 

Firth, Andrew 93 

Fischer, Jeffrey 124, 270 

Fischer, Niels 65 

Flach, Nadine 108, 197 

Flanagan, John 125, 173, 206 

Forinash, Kara 33 

Formenti, Silvia C 109 

Forne, Ignasi 283 

Forster, Nicole 159 

Fox, Paul 126, 141 

Frank, Joachim 43, 62 

Fritz, Brian 33 

Frolova, Ludmila 138, 160 

Frugier, Magali 127 

Fujiwara, Toshinobu 7 

Fukao, Akira 7 

Furman, Itay 258 

G 

Govorun, Vadim 132 

Galicia-Vazquéz, Gabriela 88 

Gallo, Simone 265 

Gamberi, Chiara 128 

Garfinkel, David 45 

Gartmann, Marco 19 

294

295 

Authors Index 

Gebauer, Fatima 37, 40 

Geggier, Peter 102 

Gehring, Niels 54, 129 

Geldreich, Angèle 44 

Genolet, Raphael 98 

Geoff, Connolly 248 

Gerber, André 230 

Giangrossi, Mara 11 

Giegé, Richard 127 

Gilbert, Robert 104, 130, 173, 206, 243 

Gilbert, Wendy 94, 252 

Gismondi, Angelo 176 

Giuliodori, Anna Maria 11 

Glickman, Michael H. 12 

Goergen, Dagmar 140 

Goetz, Christian 131 

Goldberg, Judith 109 

Goldman, Steve 231 

Golovin, Andrey 132 

Golovina, Anna 132 

Gonçalves, Juan 219 

Goodenbour, Jeffrey 103 

Goodfellow, Ian 278 

Gorgoni, Barbara 133 

Görlach, Matthias 86 

Graham, Sheila V. 82 

Grallert, Béata 155 

Gray, Nicola 70, 82, 133 

Greber, Basil 67 

Green, Rachel 62



Grela, Przemyslaw 259 

Gromeier, Matthias 41, 112, 131 

Gross, Thomas 159 

Grosshans, Helge 149 

Grosso, Stefano 134, 224 

Gruenwald, Marlene 53 

Gualerzi, Claudio 11, 61, 188 

Guarneros, Gabriel 135 

Guerbois, Mathilde 156 

Guicheney, Pascale 167 

Guo, Huili 136 

Gusmão, Leonor 212 

H 

Haas, Gabrielle 57, 137 

Haass, Christian 163, 283 

Hackmann, Alexandra 159 

Hafner, Markus 58 

Hah, Cyrus 144 

Hameete, Marleen 204 

Handelman, Samuel K. 43 

Harnisch, Christiane 197 

Harrison, Christopher B. 255 

Hartleben, Götz 193 

Hatzfeld, Mechthild 274 

Haubenreisser, Olaf 55 

Hauryliuk, Vasili 138 

Hecht, Stiven 46 

Heick Jensen, Torben 52 

Hein, Kim L. 279 

Heitzler, Domitille 196 

296

297 

Authors Index 

Helms, Sigrun 137 

Hengartner, Michael 149 

Henriksson, Niklas 202 

Hentze, Matthias 39, 54, 129, 192, 251 

Hernandez, Greco 36, 284 

Herrmann, Johannes M. 207, 216 

Herrmannova, Anna 261 

Hershey, John 239 

Heurgué-Hamard, Valérie 139 

Hill, Kirsti 237 

Hinnebusch, Alan 5, 199, 228 

Hirnet, Juliane 140 

Hochman, Tsivia 109 

Hol, Elly 231 

Holt, Christine 29 

Howard, Lyndsay 82 

Howe, Philip 141 

Hsieh, Andrew 225 

Huang, Bo 84 

Huang, Lan 256 

Huidobro-Toro, J. Pablo 175 

Humphreys, David 24 

Hung, Liang-Yi 257 

Hunt, John F. 43 

Huntsman, David 212 

Hurschler, Benjamin 149 

Hussey, George 141 

Hüttelmaier, Stefan 244 

Iadevaia, Valentina 176 

I



Igea, Ana 30 

Ignatova, Zoya 99, 122 

Igreja, Cátia 53, 57, 137, 142 

Imataka, Hiroaki 7 

Imhof, Axel 283 

Inada, Toshifumi 143 

Ingolia, Nicholas 14, 136 

Innis, C. Axel 19 

Inoue, Kunio 7 

Inouye, Masayori 282 

Irma, Dianzani 176 

Ish-Horowicz, David 35 

Itoh, Takehiko 143 

Iwakura, Nobuhiro 135 

Izaurralde, Elisa 23, 57, 142 

Jackson, Richard 150, 214, 215, 222 

Jacob, Yves 156 

Jacquemin-Sablon, Helene 283 

Jaeger, Sophie 8 

Jakovljevic, Jelena 20 

James, Laurie 227 

Jan, Eric 15, 83 

Jang, JC 144 

Jang, Sung Key 25 

Janine, Koepke 34 

Jankowska-Anyszka, Marzena 174, 202, 267, 276 

Janoskova, Martina 261 

Jansen, Ralf-Peter 205 

Jaquier-Gubler, Pascale 98 

J 

298

299 

Authors Index 

Jaroszynski, Lukasz 145 

Jean-Jean, Olivier 89 

Jemielity, Jacek 146, 157, 161, 162, 246, 251, 276, 284 

Jenner, Lasse 63 

Jennings, Martin 6 

Jinek, Martin 147 

Johansson, Marcus 84 

Jones, David 174 

Jopling, Catherine 148 

Jovanovic, Marko 149 

Juanes, Pedro P. 243 

Jungkamp, Anna-Carina 58 

Jurek, Christiane 145 

K 

Karra, Daniela 50 

Kafasla, Panagiota 150 

Kaji, Akira 135 

Kaji, Hideko 135 

Kalapala, S. 21, 69 

Kamp, Frits 163 

Kamperdick, Christine 86 

Kang, Shin Gene 144 

Kannambath, Shichina 13 

Karim, Zhala 151 

Karimian Pour, Navaz 152 

Karlsson, Stefan 176 

Kato, Yuki 143 

Kelleher, Ray 153 

Keller, Mario 44 

Kelley, Ann 64



Khorshid, Mohsen 58 

Khoshnevis, Sohail 154 

Khoury, Marie 27 

Kiebler, Michael 50 

Kieft, Jeffrey 123, 174, 213 

Kim, Yong 46 

Kimble, Judith 1 

Kimchi, Adi 170 

Kinzy, Terri 15 

Kisselev, Lev 236 

Klaholz, Bruno 60, 238 

Kleifeld, Oded 12 

Knutsen, Jon Halvor 155 

Kohler, Rebecca 67, 79 

Komarova, Anastassia 156 

Kondrashov, Alexander 280 

Kondrashov, Alexandra 95 

Kondrashov, Nadya 32 

Konevega, Andrey L. 16, 65, 188 

Konewega, Andrey 61 

Kong, YiWen 83 

König, Harald 49 

König, Julian 34 

König, Till 177 

Kononenko, Artem V. 138 

Kouba, Tomas 97 

Kowalska, Joanna 146, 157, 161, 251 

Kramer, Angela 110 

Kramer, Günter 158, 226 

Krause-Gruszczynska, Malgorzata 274 

300

301 

Authors Index 

Krebber, Heike 159 

Kremmer, Elisabeth 283 

Kress, Michel 268 

Krjuchkova, Polina 160 

Krol, Alain 165, 167 

Kropiwnicka, Anna 161 

Krushel, Les 113, 263, 269 

Kubacka, Dorota 162 

Kufel, Joanna 246 

Kühn, Hartmut 204 

Kulozik, Andreas E. 54, 129 

Kulstrunk, M. 21, 69 

Kundu, Pradipta 22 

Kuroha, Kazushige 143 

Kwon, Oh Sung 25 

La Fata, Giorgio 68 

Lacroute, François 101 

Lai, Chien-Hsien 257 

Lammich, Sven 163, 283 

Lamont, Douglas J 254 

Lamprinaki, Styliani 129 

Landthaler, Markus 58 

Lange, Vinzenz 149 

Larsson, Ola 46 

Lasko, Paul 36, 128 

Latrèche, Lynda 89 

Lawrence, Marlon 164 

Lazzaretti, Daniela 23 

Leanne, Cooper 248 

L



Leger, Melissa 42 

Lehrbach, Nic 189 

Lei, Jianlin 62 

Leichter, Michael 165 

Leigh, Kendra E. 214 

León-Avila, Gloria 135 

Lerner, Rachel 223 

Lescure, Alain 167 

Leung, Kin-Mei 168 

Leutz, Achim 169 

Levine, Paul H 109 

Li, Ka Wan 107 

Li, Xiao-Ping 259 

Liberman, Noa 170 

Liiv, Aivar 220 

Lima, Rodrigo P. 105 

Limousin, Taran 222, 241 

Lin, Chien-Ling 171 

Lin, Helen 150 

Lin, Jing-Yi 172 

Lin, Pei-Chi 144 

Lin, Zhaoru 173 

Lindahl, Lasse 164 

Lipshitz, Howard 31 

Liu, Hanqing 151 

Liu, Weizhi 174 

Lloyd, Richard 116 

Loakes, David 64 

Locker, Nicolas 227 

Lopez, Frederic 27 

302

303 

Authors Index 

Lopez-Lastra, Marcelo 175 

Lorber, Bernard 127 

Loreni, Fabrizio 176 

Lorentzen, Esben 53 

Lorsch, Jon 4, 5, 199, 232 

Lu, Jian 84 

Ludwig, Ann-Katrin 163, 283 

Luehrmann, Reinhard 3 

Luft, Eugenie 177 

Lukaszewicz, Maciej 146, 157, 161 

Lukavsky, Peter 261 

Lukoszek, Radoslaw 179 

Lunelli, Lorenzo 264 

Lutfalla, Georges 139 

Luttermann, Christine 10 

Lyabin, Dmitry 120 

Lykke-Andersen, Søren 52 

Lyons, Shawn 166 

M 

Magos-Castro, Marco Antonio 135 

Magri, Laura 265 

Maiväli, Ülo 220 

Makarov, Alexander A. 138 

Manickam, Nandini 180 

Mankin, Alexander 262 

Mann, Matthias 96 

Marchisio, Pier Carlo 134, 224, 265 

Maris, Christophe 181 

Marla, Hertz 253 

Marnef, Aline 182



Martin, Bushell 273 

Martin, Franck 8 

Martineau, Yvan 27 

Martinez-Salas, Encarna 183 

Martins, Rafael M. 228 

Maruyama, Risa 271 

Marzi, Stefano 60, 238 

Marzluff, William 166, 223 

Mateus, Denisa 184 

Mateyak, Maria M. 15 

Mathys, Hansruedi 110 

Matic, Ivan 96 

Matt, T. 21 

Mattioli, Claudia 68 

Mauxion, Fabienne 185 

Mayer, Matthias P. 226 

Mc Mahon, Robert 186 

McCarthy, John E.G. 13, 104, 243 

McCracken, Lora 70 

McFarland, Craig 174 

McGivern, Jered 33 

Mechulam, Yves 201 

Meijer, Hedda 106 

Mendes, Filipa 219 

Mendes, Pedro 13 

Méndez, Raúl 30 

Merenbakh, Keren 217 

Merrick, William 28 

Mestel, Celine 187 

Meyers, Gregor 10 

304

305 

Authors Index 

Meyuhas, Oded 225 

Michael H. W., Weber 260 

Michael, Rist 248 

Mielke, Thorsten 19 

Mihailovich, Marija 40 

Mikl, Martin 50 

Milon, Pohl 61, 188 

Miluzio, Annarita 224 

Minshall, Nicola 162 

Miron, Mathieu 36 

Miska, Eric 26, 149, 189 

Mitkevich, Vladimir A. 138 

Mohammad-Qureshi, Sarah 190 

Mokrejš, Martin 191 

Mollet, Stéphanie 268 

Monfort-Prieto, Elena 271 

Mongeard, Rémi 139 

Moon, Alice 106 

Moraes, Maria Carolina S. 228 

Moretti, Francesca 192 

Morgner, Nina 150 

Moritz, Bodo 197 

Morley, Simon 272 

Morris, Feldman 225 

Morth, J. Preben 279 

Motz, Carina 57 

Moura, Danielle M. N. 105 

Mühlemann, Oliver 52 

Müller, Christine 193 

Müller, Marisa 205



Munro, James 17 

Munschauer, Mathias 58 

Munzarova, Vanda 194 

Musner, Nicolo' 195 

Musnier, Astrid 196 

N 

Naarmann, Isabel 197 

Nadera, Ainaoui 198 

Nag, Nabanita 180 

Nakamura, Yoshikazu 121 

Namy, Olivier 125 

Nanda, Jagpreet 5, 199 

Napthine, Sawsan 200 

Natasha, Webb 248 

Naveau, Marie 201 

Navid, Sadri 48 

Navon, Sivan 258 

Nelson, Bradley 123 

Netzer, Nir 103 

Neumann, Beate 87 

Neu-Yilik, Gabriele 54 

Niedzwiecka, Anna 174, 202 

Niepmann, Michael 140, 203 

Nieradka, Andrzej 204 

Nierhaus, Knud H. 151 

Niessing, Dierk 205 

Nieto, Amelia 278 

Nikolic, Emily 206 

Nilsson, Jakob 279 

Nilsson, Per 202 

306

307 

Authors Index 

Nissen, Poul 279 

Nousch, Marco 242 

Nuscher, Brigitte 163 

O 

Ohlmann, Theo 222, 241 

Olinger, Helga 49 

Oliveira, Carla 212 

Oliveira, Patrícia 212 

Olsen, Jesper 96 

Ortiz, Pedro 15 

Ortiz-Meoz, Rodrigo F. 62 

Ostareck, Dirk H. 108, 197 

Ostareck-Lederer, Antje 108, 197 

Ott, Martin 207, 216 

Ovchinnikov, Lev 120 

Ozgur, Sevim 56 

Pech, Markus 151 

Pacheco, Almudena 183 

Pacheco, Teresa R 110 

Paek, Ki Young 25 

Pain, Jenny 272 

Paleskava, Alena 16 

Pan, Tao 103 

Panek, Josef 194 

Papadopoulou, Barbara 254 

Park, Ji Hoon 25 

Passmore, Lori 232 

Pavesi, Giulio 40 

P



Pavitt, Graham 6, 190 

Pederzolli, Cecilia 264 

Peil, Lauri 220 

Pelletier, Jerry 88 

Perez-Martinez, Xochitl 208 

Pesce, Elisa 134 

Peterson, Mark 77 

Pfeilschifter, Josef 114 

Phillips, Nicola 209 

Picotti, Paola 149 

Pierre, Philippe 210 

Pierron, Gérard 268 

Pilar, Martin-Marcos 199 

Pilotte, Julie 211 

Pilpel, Yitzhak 258 

Pineiro, David 183 

Pinheiro, Hugo 212 

Piper, M 29 

Plank, Terra-Dawn 213 

Pohlmann, Thomas 34 

Polacek, Chanti 76 

Polacek, Norbert 92 

Pollard, Hilary 272 

Polunovsky, Vitaly 46 

Pomeranz, Marcelo 144 

Pon, Cynthia 11 

Porco Jr, John A 88 

Pospisek, Martin 191, 266 

Postma, Nienke 231 

Poupon, Anne 196 

308

309 

Authors Index 

Powell, Michael 214 

Poyry, Tuija 150, 214, 215, 222 

Prats, Anne-Catherine 27 

Preiss, Thomas 24, 251 

Prestele, Martin 207, 216 

Prinzen, Claudia 163 

Prizant, Maya 217 

Procaccianti, Claudio 134 

Prongidi-Fix, Lydia 8 

Prouteau, Manoel 101 

Provenzani, Alessandro 218, 264 

Pulk, Arto 220 

Pusic, Aya 32 

Pyronnet, Stephane 27 

Q 

Quattrone, Alessandro 218, 264 

R 

Raabe, Monika 68 

Rajiv, Khanna 248 

Rakauskaite, Rasa 55 

Ramajo, Jorge 183 

Ramakrishnan, V 64, 232 

Ramalho, Anabela 219 

Ramanathan, Preethi 81 

Ramdohr, Pablo 175 

Ramu, Haripriya 262 

Ranzato, Elia 134 

Rasmussen, Thomas Bruun 76 

Re, Angela 218



Rederstorff, Mathieu 167 

Reineke, Lucas 28 

Reis, Christian R. S. 105 

Reiter, Eric 196 

Reiter, Lukas 149 

Remme, Jaanus 220 

Rhoads, Robert 47 

Ribeiro, Luís 221 

Ricci, Emiliano P. 222, 241 

Ricciardi, Adele 223 

Richard, Pascale 167 

Richardson, William 133 

Ringel, Inbal 35 

Robaire, Bernard 277 

Roberts, Lisa 76 

Robinson, Carol 150 

Rodnina, Marina 16, 61, 65, 188 

Rodriguez, Felipe E. 175 

Romby, Pascale 60 

Rosenblum, Kobi 38 

Rothballer, Andrea 58 

Ruggeri, Valentina 224 

Ruggero, Davide 225 

Rutkai, Edit 261 

Ryabova, Lyubov 44 

Rydzik, Anna 146 

Ryu, Incheol 25 

Serebryakova, Marina 132 

Saini, Adesh 5, 199 

S 

310

311 

Authors Index 

Sakamoto, Hiroshi 7 

Salaun, Christine 82 

Sanbonmatsu, Kevin 17 

Sandikci, Arzu 226 

Santos, Manuel 184 

Santos, Sandra 221 

Sargueil, Bruno 227 

Sasano, Yumi 7 

Sato, Brian 271 

Sattlegger, Evelyn 228 

Saunders, William 20 

Sawicka, Kirsty 273 

Schaeffer, Laure 8 

Schaffitzel, Christiane 67, 79 

Scheckel, Claudia 229 

Schenk, Luca 230 

Schenkman, Sergio 254 

Scheper, Gert 231 

Schepetilnikov, Mikhail 44 

Schleicher, Ulrike 177 

Schmandke, Antonio 168 

Schmeing, T. Martin 232 

Schmitt, Emmanuelle 201 

Schneider, Robert 48, 109, 187 

Schreiber, Sandra 193 

Schreiner, Eduard 233, 255 

Schrimpf, Sabine 149 

Schubert, Mario 118 

Schulten, Klaus 19, 62, 255 

Schuman, Erin 85



Sehr, Peter 87 

Seidelt, Birgit 19 

Seixas, Susana 212 

Séraphin, Bertrand 101, 185 

Sergiev, Petr 132 

Seruca, Raquel 212 

Sha, Zhe 12 

Shapira, Michal 42 

Sharma, Pamila 115 

Shatsky, Ivan 71, 203, 250 

Shchepetilnikov, Mikhail 234 

Sheets, Michael 33, 133 

Sherbakov, D. 21 

Shields, Michael 124, 270 

Shih, Shin-Ru 172 

Shin, Nara 235 

Shirashige, Katsuhiko 143 

Shiroishi, Toshihiko 32 

Shirokikh, Nikolay 236 

Shokat, Kevan 225 

Shveygert, Mayya 112 

Sicheri, Frank 111 

Simmonds, Rachel 237 

Simonetti, Angelita 238 

Slevin, Michael 166 

Smales, Mark 75 

Small, Pamela 237 

Smink, Jeske 169 

Smit, August B. 107 

Smith, Corey 248 

312

313 

Authors Index 

Smith, Paul C. 43 

Sokabe, Masaaki 239 

Sonenberg, Nahum 7, 36, 147, 277, 284 

Soppa, Joerg 240 

Soto Rifo, Ricardo 222, 241 

Souquere, Sylvie 268 

Spahn, Christian 66 

Spirin, Alexander 236 

Spriggs, Keith 80, 95, 273 

Spriggs, Ruth 75, 273 

Springer, Mathias 60 

Squires, Jeffrey 242 

Stalder, Lukas 52 

Standart, Nancy 26, 162, 182 

Stark, Holger 65 

Steitz, Thomas 19 

Stepinksi, Janusz 174, 267 

Stepinski, Janusz 161, 162, 276 

Stevenson, Abigail 243 

Stoecklin, Georg 56 

Stöhr, Nadine 244 

Stolarski, Ryszard 174 

Strader, Michael Brad 103 

Straesser, Katja 96 

Strein, Claudia 39 

Strenkowska, Malwina 146 

Subramanian, A. 21, 69 

Sukarieh, Rami 88 

Sun, Lijie 104 

Superti-Furga, Giulio 50



Suresh, Susmitha 45 

Susmitha, Suresh 245 

Suter, Catherine 242 

Szczepaniak, Sylwia 246 

Tabas, Ira 275 

Tahiri-Alaoui, Abdessamad 247 

Takacs, Julie 199 

Tang, Anna 115 

Tangy, Frederic 156 

Tapia, Karla 175 

Tavares-Carreon, Faviola 208 

Tchorzewski, Marek 259 

Tebaldi, Toma 218 

Tellam, Judy 248 

Temme, Claudia 249 

Tenson, Tanel 138 

Terenin, Ilya 71, 203 

Terry, Daniel 102 

Tesseraud, Sophie 196 

Tettweiler, Gritta 36 

Thermann, Rolf 192, 251 

Thiébeauld, Odon 44 

Thoma, Christian 7, 87 

Thomas, Preiss 242 

Thompson, Mary K. 252 

Thompson, Sunnie 253 

Tim, Gant 273 

Toledo, Juliano 254 

Tony, Vuocolo 248 

T 

314

315 

Authors Index 

Totaro, Antonio 68 

Tournier, Isabelle 23 

Trabuco, Leonardo 19, 62 

Tritschler, Felix 23, 57 

Truffault, Vincent 23, 57 

Tsai, Becky 256 

Tseng, Joseph Ta-Chien 257 

Tuck, Simon 90 

Tuller, Tamir 258 

Tumer, Nilgun 259 

Tung, Chang-Shung 17 

Tung, YCL 29 

Tuschl, Thomas 58 

Tvegård, Tonje 155 

Tyler, Rebecca 190 

U 

Ufer, Christopher 204 

Ulrich, Alexander 58 

Ulryck, Nathalie 227 

Umesh, Varshney 260 

Underwood, Jon 46 

Urlaub, Henning 68, 197 

Valasek, Leos 97, 194, 261 

Valiente-Echeverría, Fernando 175 

Vallejos, Maricarmen 175 

van der Knaap, Marjo 231 

van Kollenburg, Barbara 231 

Vanderklish, Peter 211 

V



Vardimon, Lily 217 

Vasella, A. 21 

Vassilenko, Konstantin 236 

Vazquez-Laslop, Nora 262 

Vendra, Georgia 50 

Veo, Bethany 267 

Vestsigian, Kalin 258 

Vidalain, Pierre-Olivier 156 

Viero, Gabriella 218, 264 

Villlalba, Ana 37 

Virtanen, Anders 202 

Vivanco-Domínguez, Serafín 135 

Vlasak, Markete 272 

Vogt, Peter H 145 

Volta, Viviana 265 

von Lindern, Marieke 204 

Von Weymarn, Linda 46 

Voorhees, Rebecca 64 

Vopalensky, Vaclav 266 

W 

Wagner, Gerhard 42 

Wagner, Susan 261 

Wahle, Elmar 249 

Wallace, Adam 267 

Wang, Xi 13 

Wang, Xiaorong 256 

Wasserman, Michael 17 

Waterman, Marian 256 

Webb, Thomas 80 

Weichenrieder, Oliver 57 

316

317 

Authors Index 

Weil, Dominique 268 

Weinlich, Susan 108 

Weiss, Manuel 149 

Weissman, Jonathan 14, 136 

Westerhoff, Hans 13 

Wethmar, Klaus 169 

Wickens, Marvin 33 

Wie, Sten 269 

Wieden, Hans-Joachim 124, 270 

Wilczynska, Ania 26 

Wilhelm, Jim 271 

Wilkie, Gavin 133 

Willcocks, Margaret 76 

Willett, Mark 272 

Willis, Anne 75, 83, 95, 237, 273, 280 

Wilson, Daniel 19 

Wilson, Lindsay 95 

Wintermeyer, Wolfgang 61, 65 

Wolf, Annika 274 

Wolf, Dieter 12 

Wong, Ying Ying 106 

Woo, Connie Wai Hong 275 

Woolford, John 20 

Worch, Remigiusz 202 

Wrabetz, Lawrence 195 

Wurth, Laurence 165 

Wypijewska, Anna 276 

Yamaguchi, Yoshihiro 282 

Yan, Fu 115 

Y



Yanagiya, Akiko 277 

Yángüez, Emilio 278 

Yates, Luke 130 

Yatime, Laure 279 

Yeo, GSH 29 

Yewdell, Jonathan W. 103 

Yoffe, Yael 42 

Young, Lucy 280 

Yusupov, Marat 63, 238 

Yusupova, Gulnara 63 

Zaborowska, Izabela 281 

Zambelli, Federico 40 

Zambroni, Desirèe 195 

Zamudio-Ochoa, Angelica 208 

Zannat, Thangima 78 

Zarnack, Kathi 34 

Zavolan, Mihaela 58 

Zdanowicz, Agnieszka 251 

Zekri, Latifa 23 

Zengel, Janice 164 

Zhang, Gong 122 

Zhang, Lianbing 249 

Zhang, Linda 131 

Zhang, Wen 59, 117 

Zhang, Yan 33 

Zhang, Yonglong 282 

Zhao, Rui 174 

Zhou, Jia 55 

Zilow, Sonja 163, 283 

Z 

318

319 

Authors Index 

Zimmer, Jutta 145 

Zinoviev, Alexandra 42 

Zivrai, KH 29 

Zolfaghari, Ladan 109 

Zollo, Ornella 225 

Zuberek, Joanna 42, 146, 157, 161, 162, 246, 284

A 

TILMANN ACHSEL 

VIB - K.U. Leuven 

Herestraat 49 bus 602 

3001 Leuven 

Belgium 

Tilmann.Achsel@med.kuleuven.be 

SARAH ADIO 

MPI Biophysical Chemistry, Göttingen 

Am Faßberg 11 

37077 Göttingen 

Niedersachsen 

Germany 

Sarah.Adio@mpibpc.mpg.de 

RASHID AKBERGENOV 

Institute of Medical Microbiology, 

University of Zurich 

Gloriastrasse 32 

8006 Zurich 

Switzerland 

rakbergenov@imm.uzh.ch 

CHRISTINE ALLMANG 

Université de Strasbourg, CNRS 

IBMC, 15 rue René Descartes 

67084 Strasbourg 

France 

c.allmang@ibmc.u-strasbg.fr 

321 

List of Participants 

ROSS ANDERSON 

MRC Human Reproductive Sciences 

Unit, University of Edinburgh 

Centre for Reproductive Biology 

EH16 4TJ Edinburgh 

United Kingdom 

r.anderson2@hrsu.mrc.ac.uk 

DMITRY ANDREEV 

Moscow State University 

Khokhlov street 

119992 Moscow 

Russian Federation 

cycloheximide@yandex.ru 

JOSHUA ARRIBERE 

MIT - Gilbert Lab 

143 Albany St Apt 319B 

02139 Cambridge, Massachusetts 


arribere@mit.edu 

B 

MICHELLE BADURA 

NYU Medical Center 

550 1st Avenue 

10016 New York, New York 


mlb387@med.nyu.edu


EMBL Heidelberg, 9–13 September 2009 

VIDYA BALAGOPAL 

University of Arizona 

1007 E Lowell Street, #403 

85721 Tucson, Arizona 


vidya@email.arizona.edu 

MARIA BARNA 

University of California, San 

Francisco 

Mission Bay Campus 

94158 San Francisco, California 


maria.barna@ucsf.edu 

DAVID BARTEL 

MIT/Whitehead Institute/HHMI 

Whitehead Institute 

02445 Cambridge, MA 


dbartel@wi.mit.edu 

KRISTEN BARTOLI 

University of Pittsburgh, School of 

Medicine 

4249 5th Ave 

15260 Pittsburgh, Pennsylvania 


kristen.bartoli@gmail.com 

322 

AMANDINE BASTIDE 

University of Nottingham 

Centre for Biomolecular Sciences 

NG7 2RD UK Nottingham 


amandine.bastide@nottingham.ac.uk 

SEBASTIAN BAUMANN 

Max Planck Institute for Terrestrial 

Microbiology 

Karl von Frisch Strasse 

35043 Marburg 

Germany 

baumannb@mpi-marburg.mpg.de 

GRAHAM BELSHAM 

Technical University of Denmark 

Lindholm 

4771 Kalvehave 

Denmark 

grbe@vet.dtu.dk 

ALEXEY BENYUMOV 

Department of Medicine, University of 

Minnesota 

#387 KE, 425 East River Rd. 

55455 Minneapolis, Minnesota 


benyu004@umn.edu

MARLA BERRY 

University of Hawaii 

651 Ilalo Street 

93618 Honolulu, Hawaii 


mberry@hawaii.edu 

RUMPA BHATTACHARJEE 

University of Guelph 

50 Stone Road E 

N1G 2W1 Guelph 

Ontario, Canada 

rbiswas@uoguelph.ca 

SHASHI BHUSHAN 

Gene Cemter, LMU Munich 

Feodor-Lynen-Str. 25 

81377 Munich 

Germany 

bhushan@lmb.uni-muenchen.de 

STEFANO BIFFO 

Fondazione Centro San Raffaele del 

Monte Tabor & Università del 

Piemonte Orientale 

Via Olgettina 58 

20132 Milano 

Italy 

biffo.stefano@hsr.it 

323 


SCOTT BLANCHARD 

Weill Cornell Medical College 

1300 York Avenue 



scb2005@med.cornell.edu 

DANIEL BOEHRINGER 

ETH 

Schafmattstr. 20 

8093 Zurich 


boehringer@mol.biol.ethz.ch 

GREGORY BOEL 

Columbia University 

702A Fairchild Center 



gb2186@columbia.edu 

FULVIA BONO 

Max Planck of Dev. Biology 

Spemannstrasse 35 

72076 Tuebingen 

Germany 

fulvia.bono@tuebingen.mpg.de 

ERIK BÖTTGER 

Institute of Medical Microbiology 


8006 Zürich 


boettger@imm.uzh.ch



ANDREW BOTTLEY 

The University of Nottingham 

University Park 

NG7 2RD Nottingham 


andrew.bottley@nottingham.ac.uk 

SHELTON BRADRICK 

Duke University 

Department of Molecular Genetics 

and Microbiology 

27710 Durham, North Carolina 


shelton.bradrick@duke.edu 

IAN BRIERLEY 

University of Cambridge 

Tennis Court Road 

CB2 1QP Cambridge 


ib103@mole.bio.cam.ac.uk 

SAVERIO BROGNA 

University of Birmingham 

Edgbaston 

B15 2TT Birmingham 


s.brogna@bham.ac.uk 

HANNAH BURGESS 

MRC HRSU / University of Edinburgh 

Queen's Medical Research Institute 

EH164TJ Edinburgh 


hannah.burgess@hrsu.mrc.ac.uk 

324 

MARTIN BUSHELL 


Centre For Biomolecular Sciences 



martin.bushell@nottingham.ac.uk 

ANDERS BYSTRÖM 

Umea University 

Build-6L 

901 87 Umea 

Sweden 

anders.bystrom@molbiol.umu.se 

C 

IVÁN CAJIGAS 

California Institute of Technology 

HHMI & Division of Biology 

91125 Pasadena 

California 


icajigas@caltech.edu 

CORNELIS CALKHOVEN 

Leibniz Institute for Age Research - 

Fritz Lipmann Institute 

Beutenbergstr. 11 

07745 Jena 

Germany 

calkhoven@fli-leibniz.de

CLAUDIA CASANOVA 

University Hospital of Freiburg - 

EMBL/Heidelberg 

Hugstetterstr. 55 

79106 Freiburg 

Germany 

claudia.casanova@uniklinikfreiburg.de 

BEATRIZ CASTILHO 

UNIFESP 

Rua Botucatu, 862 

04023-062 Sao Paulo 

Brazil 

bacastilho@unifesp.br 

JAMIE CATE 

UC Berkeley 

708B Stanley Hall 

94720 Berkeley, California 


jcate@lbl.gov 

REGINA CENCIC 

McGill University 

Department of Biochemistry 

H3G 1Y6 Montreal 

Quebec, Canada 

regina.cencic@mcgill.ca 

325 

LAURENT CHAVATTE 


CNRS 

Centre de Génétique Moléculaire 

91198 Gif-sur-Yvette Cedex 

France 

chavatte@cgm.cnrs-gif.fr 

CHANGCHUN CHEN 

Umeå university 

Build 6L 

901 87 Umea 

Sweden 

changchun.chen@molbiol.umu.se 

WEI WEN CHIEN 

Université Claude Bernard Lyon 1, 

CNRS UMR 5239 

Faculté de Médecine Lyon Sud 

69921 Oullins 

France 

weiwen86@gmail.com 

ANNA CHIRKOVA 

Innsbruck Medical University, 

Biocenter 

Fritz-Pregl-Str.3 

6020 Innsbruck 

Austria 

anna.chirkova@i-med.ac.at



MYUNG-HAING CHO 

Seoul National University 

College of Veterinary Medicine 

151-742 Seoul 

Korea, Republic of 

mchotox@snu.ac.kr 

BETTY CHUNG 

Recoding Lab 

Na Cork 

Ireland 

betty.yingwen.chung@gmail.com 

BRYAN CLARKSON 

University of California, Berkeley 

731 Stanley Hall 



bryan_clarkson@berkeley.edu 

LAURA COBBOLD 



NG72RD Nottingham 


laura.cobbold@nottingham.ac.uk 

ELENA CONTI 

Max-Planck-Institut für Biochemie 

Am Klopferspitz 18 

D-82152 Martinsried 

Germany 

conti@biochem.mpg.de 

326 

BRITTA COORDES 

LMU Munich 

Feodor-Lynen-Straße 25 

81377 München 

Germany 

coordes@lmb.uni-muenchen.de 

BERTRAND COSSON 

Université Pierre et Marie Curie 

Place Georges Tessier 

29680 Roscoff 

France 

cosson@sb-roscoff.fr 

LUCIE CUCHALOVA 

Academy of Sciences of the Czech 

Republic, Institute of Microbiology 

Videnska 1083 

142 20 Prague 


cuchalova@biomed.cas.cz 

JOSEPH CURRAN 

University of Geneva Medical School 

(CMU) 

1 rue Michel Servet 

1206 Geneva 


Joseph.Curran@unige.ch

ANDREAS CZECH 

University Potsdam 

Lindenstraße 25 

14467 Potsdam 

Germany 

aczech@uni-potsdam.de 

D 

EDWARD DARZYNKIEWICZ 

Warsaw University 

ul. Zwirki i Wigury 93 

02-089 02-089 Warsaw 

Poland 

edek@biogeo.uw.edu.pl 

MARIE-CLAIRE DAUGERON 

Equipe labellisée La Ligue, CGM – 

CNRS, avenue de la Terrasse 

91198 Gif sur Yvette Cedex, France 

and Université Paris Sud XI 

1 avenue de la terrasse 

91190 Gif sur Yvette 

France 

daugeron@cgm.cnrs-gif.fr 

RICHA DAVE 


420 East 70th Street Apt. 8M 

10021 New York City 

New York 


rd227@cornell.edu 

327 

ALEXANDRE DAVID 

NIH 

Bldg 33, 33 North Drive 

20892 Bethesda 

Maryland 


davidal@niaid.nih.gov 

RICHARD DAVIS 


University of Colorado School of 

Medicine 

80010 Aurora 

Colorado 


richard.davis@ucdenver.edu 

LUIGI DE COLIBUS 

Oxford University 

The Henry Wellcome Building for 

Genomic Medicine 

OX3 7BN Oxford 


luigi@strubi.ox.ac.uk 

OSVALDO DE MELO NETO 

Centro de Pesquisas Aggeu 

Magalhães / Fundação Oswaldo Cruz 

Avenida Moraes Rego, s/n 

50670-420 Recife, Pernambuco 

Brazil 

opmn@cpqam.fiocruz.br



CORNELIA DE MOOR 





cornelia.demoor@nottingham.ac.uk 

SILVIA DE RUBEIS 

Flanders Institute for Biotechnology 

(VIB) 

Rijvisschestraat 120 

9052 Zwijnaarde 

Belgium 

silvia.derubeis@student.kuleuven.be 

MAARTEN DE SMIT 

Leiden Institute of Chemistry 

Einsteinweg 55 

2333 CC Leiden 

Netherlands 

m.smit@chem.leidenuniv.nl 

SEBASTIAN DE VRIES 

Martin-Luther University Halle- 

Wittenberg 

Kurt-Mothes-Str. 3 

06120 Halle (Saale) 

Germany 

sebastian.de-vries@biochemtech.unihalle.de 

328 

SILVERA DEBORAH 

NYU School of Medicine 

550 1st Ave 



deborah.silvera@nyumc.org 

NATALIA DEMESHKINA 

IGBMC 

BP 10142 

67404 Illkirch 

France 

natidem@igbmc.fr 

JOANA DESTERRO 

Institute Molecular Medicine 

Edificio Egas Moniz 

1649-028 Lisboa 

Portugal 

joanadesterro@fm.ul.pt 

THOMAS DEVER 

NIH 

6 Center Dr 

20892 Bethesda, Maryland 


tdever@nih.gov 

RIVKA DIKSTEIN 

Weizmann Institute of Science 

2 Hertzl St. 

76100 Rehovot 

Israel 

rivka.dikstein@weizmann.ac.il

JONATHAN DINMAN 

University of Maryland 

Dept. Cell Biology & Molecular 

Genetics 

20742 College Park, Maryland 


dinman@umd.edu 

SERGEY DMITRIEV 

Belozersky Institute of Physico- 

Chemical Biology 

GSP-1, Leninskie Gogy, MSU, Bldg. 

"A" 

119991 Moscow 


dmitriev_sergey@genebee.msu.su 

ELENA DOBRIKOVA 

Duke University Medical Center 

Sands Bldg., r.204 



dobri001@mc.duke.edu 

TARA DOBSON 

University of Colorado 

12801 East 17th Avenue 

80045 Aurora, Colorado 


tara.dobson@ucdenver.edu 

329 

ANKE DOLLER 


Hospital of the J. W. Goethe 

University 

Theodor-Stern-Kai 7 

60590 Frankfurt 

Germany 

a.doller@med.uni-frankfurt.de 

VICTORIA DORONINA 

Newcastle University 

Institute for Cell and Molecular 

Biosciences 

NE2 4HH Newcastle upon Tyne 


mstislavl@hotmail.com 

JONATHAN DOUGHERTY 

Baylor College of Medicine 

One Baylor Plaza 

77030 Houston, Texas 


doughert@bcm.edu 

KENT DUNCAN 

EMBL 

Meyerhofstrasse 1 

69117 Heidelberg 

Germany 

duncan@embl.de



JACK DUNKLE 

UC-Berkeley 




dunkle@berkeley.edu 

OLIVIER DUSS 

ETH Zurich 

Schafmattstrasse 20, HPK G9.3 

8093 Zurich 


oduss@mol.biol.ethz.ch 

E 

CHRISTIAN ECKMANN 

MPI-CBG 

Pfotenhauerstrasse 108 

01307 Dresden 

Germany 

eckmann@mpi-cbg.de 

NAAMA ELDAD 

Technion 

Faculty of Medicine, 

31096 Haifa 

Israel 

naamael@tx.technion.ac.il 

IRINA ELISEEVA 

Institute of Protein Research 

Institutskaya str. 4 

142290 Pushchino 


eliseevaia@rambler.ru 

330 

KEI ENDO 

Institute of Medical Science, 

University of Tokyo 

4-6-1 Shirokanedai, Minato-ku 

108-8639 Tokyo 

Japan 

k_and_o@ims.u-tokyo.ac.jp 

ANNE EPHRUSSI 

EMBL Heidelberg 

Meyerhofstr. 1 


Germany 

ephrussi@embl.de 

F 

PHILIP FARABAUGH 

University of Maryland Baltimore 

County 

21250 Baltimore, Maryland 


farabaug@umbc.edu 

IVAN FEDYUNIN 

Potsdam University 

Zeppelinstr., 45 

14471 Potsdam 

Germany 

fedyunin@uni-potsdam.de

MEGAN FILBIN 

University of Colorado Denver, 

Anschutz Medical Campus 

Biochemistry and Molecular Genetics 



megan.filbin@ucdenver.edu 

WITOLD FILIPOWICZ 

Friedrich Miescher Institute for 

Biomedical Research 

Maulbeerstrasse 66 

4058 Basel 


Witold.Filipowicz@fmi.ch 

HELENA FIRCZUK 

Manchester Interdisciplinary 

Biocentre 

M1 7DN Manchester 


helena.firczuk@manchester.ac.uk 

JEFFREY FISCHER 

University of Lethbridge 

#7 Columbia Place 

T1K 5A8 Lethbridge 

Alberta, Canada 

jeffrey.fischer@uleth.ca 

331 


NIELS FISCHER 

Max Planck Institute for Biophysical 

Chemistry 

37077 Goettingen 

Germany 

niels.fischer@gmx.de 

JOHN FLANAGAN 

University of Oxford 



john@strubi.ox.ac.uk 

PAUL FOX 

Cleveland CLinic 

Dept. Cell Biology 

44195 Cleveland, Ohio 


foxp@ccf.org 

JOACHIM FRANK 

HHMI, Columbia University 

650 West 168th Street 



jf2192@columbia.edu 

MAGALI FRUGIER 

UPR ARN du CNRS 

IBMC 


France 

m.frugier@ibmc.u-strasbg.fr



TOSHINOBU FUJIWARA 

Kobe University 

1- 1 Rokkodai, Nada 

657-8501 Kobe 

Japan 

tosinobu@kobe-u.ac.jp 

AKIRA FUKAO 

Kobe University 

1-1 rokkodaicyo nadaku 

657-8501 Kobe 

Japan 

fukaokun@kobe-u.ac.jp 

G 

CHIARA GAMBERI 


1205 Dr Penfield Avenue 

H3A 1B1 Montreal 


chiara.gamberi@mcgill.ca 

FATIMA GEBAUER 

Fundacio Privada Centre De 

Regulacio Genomica 

Dr. Aiguader, 88, 6TH Floor 

08003 Barcelona 

Spain 

fatima.gebauer@crg.es 

332 

NIELS GEHRING 

EMBL 



Germany 

gehring@embl.de 

ROBERT GILBERT 

University of Oxford 

Wellcome Trust Centre for Human 

Genetics 



gilbert@strubi.ox.ac.uk 

WENDY GILBERT 

MIT 

31 Ames St. 



wgilbert@mit.edu 

GENEVIÈVE GIRARD 

Universiteit Leiden 


2333CC Leiden 


girardgao@chem.leidenuniv.nl

ANNA MARIA GIULIODORI 

Laboratory of Genetics 

Department of Biology MCA 

University of Camerino 

via Gentile III da Varano 

62032 Camerino 

Italy 

annamaria.giuliodori@unicam.it 

CHRISTIAN GOETZ 

Duke University 

100 Village Circle Way 



cg33@duke.edu 

ANNA GOLOVINA 


Russia, Moscow, Leninskie Gory 

street 

119992 Moscow 


malanka@yandex.ru 

BARBARA GORGONI 

MRC-Human Reproductive Sciences 

Unit 



b.gorgoni@hrsu.mrc.ac.uk 

333 

NICOLA GRAY 

Medical Research Council 

QMRI 



n.gray@hrsu.mrc.ac.uk 

STEFANO GROSSO 


DIBIT, San Raffaele Scientific 

Institute 

via Olgettina 58 

20132 Milan 

Italy 

grosso.stefano@hsr.it 

CLAUDIO GUALERZI 

University of Camerino 

via Gentile III da Varano 

62032 Camerino 

Italy 

claudio.gualerzi@unicam.it 

GABRIEL GUARNEROS 

CINVESTAV 

Av. Instituto Politécnico Nacional 

2508 

07360 Mexico City 

Mexico 

gguarner@cinvestav.mx



HUILI GUO 

Whitehead Institute for Biomedical 

Research 

Department of Biology, 

Massachusetts Institute of 

Technology 

9 Cambridge Center 



hguo@mit.edu 

VADIM GURVICH 

University of Minnesota 

717 Delaware St. SE 



vadimg@umn.edu 

H 

GABRIELLE HAAS 

Max Planck Institute for 

Developmental Biology 



Germany 

gabrielle.haas@tuebingen.mpg.de 

VASILI HAURYLIUK 

University of Tartu, Institute of 

Technology 

Nooruse St 1, Room 400 

50411 Tartu 

Estonia 

vasili.hauryliuk@ut.ee 

334 

MATTHIAS HENTZE 




Germany 

hentze@embl.de 

GRECO HERNANDEZ 


1205 Dr. Penfield Ave. 

QC H3A 1B1 Montreal 


greco.hernandez@mcgill.ca 

VALÉRIE HEURGUÉ-HAMARD 

CNRS, IBPC 

IBPC, CNRS UPR9073 

75005 Paris 

France 

heurgue@ibpc.fr 

ALAN HINNEBUSCH 

National Institutes of Health 

Building 6, Room 230 

20892 Bethesda, Maryland 


ahinnebusch@nih.gov 

JULIANE HIRNET 

Justus Liebig University 

Friedrichstr. 24 

35392 Giessen 

Germany 

jhirnet@gmail.com

CHRISTINE HOLT 


Anatomy Building 

CB2 3DY Cambridge 


ceh@mole.bio.cam.ac.uk 

PHILIP HOWE 

Cleveland Clinic 

The Lerner Research Institute 

44195 Cleveland, Ohio 


howep@ccf.org 

I 

CÁTIA IGREJA 

MPI for Developmental Biology 

Tuebingen 

Spemannstr. 35 

D-72076 Tuebingen 

Germany 

catia.igreja@tuebingen.mpg.de 

TOSHIFUMI INADA 

Nagoya University 

Chikusaku Furocyo 

46408602 Nagoya 

Japan 

p47294a@nucc.cc.nagoya-u.ac.jp 

335 

NICHOLAS INGOLIA 


University of CaliforniaByers Hall 

Room 403, UCSF MC 2542 

94143 San Francisco, California 


ingolia@cmp.ucsf.edu 

C. AXEL INNIS 

Yale University 

Bass 415, 266 Whitney Avenue 

06520 New Haven 

Connecticut 


axel.innis@yale.edu 

ELISA IZAURRALDE 

Max Planck Institute for 




Baden-Wuerttemberg 

Germany 

elisa.izaurralde@tuebingen.mpg.de 

J 

SUNG KEY JANG 

POSTECH 

Molecular Virology Laboratory, PBC, 

Pohang University of Science and 

Technology, San31, Hyoja-dong 

790-784 Pohang 

Republic of Koreo 

sungkey@postech.ac.kr



JC JANG 

Ohio State University 

43210 Columbus 

Ohio 


jang.40@osu.edu 

LUKASZ JAROSZYNSKI 

University Women Hospital 

Voßstrasse 9 


Germany 

lukasz.jaroszynski1@googlemail.com 

JACEK JEMIELITY 

University of Warsaw 

ul. Krakowskie Przedmiescie 26/28 

00-927 Warsaw 


jacekj@biogeo.uw.edu.pl 

LASSE JENNER 

CERBM-GIE / IGBMC 

1, rue Laurent Fries 


France 

lasse@igbmc.u-strasbg.fr 

MARTIN JENNINGS 

University of Manchester 

M13 9PT Manchester 


martin.jennings-3@manchester.ac.uk 

336 

MARTIN JINEK 

University of California, Berkeley 


94709 Berkeley 


jinek@berkeley.edu 

CATHERINE JOPLING 


University Park 



catherine.jopling@nottingham.ac.uk 

MARKO JOVANOVIC 


Winterthurerstrasse 190 

8057 Zurich 


marko.jovanovic@molbio.uzh.ch 

K 

PANAGIOTA KAFASLA 


80 Tennis Court road 

CB2 1GA Cambridge 


pk303@cam.ac.uk

AKIRA KAJI 

University of Pennsylvania 

225 Johnson Pavilion 

19104-6076 Philadelphia 

Pennsylvania 


kaji@mail.med.upenn.edu 

ZHALA KARIM 

Max Planck Institute für Molekulär 

Genetik 

Ihnestrasse 63-73 

14195 Berlin 

Germany 

karim@molgen.mpg.de 

NAVAZ KARIMIAN POUR 

University of Toronto 

Room 3403 

M5G 1X8 Toronto 

Ontario, Canada 

navazibb@gmail.com 

RAY KELLEHER 

Harvard Medical School 

MGH-Simches Research Center 

02114 Boston 

Massachusetts 


kelleher@helix.mgh.harvard.edu 

337 

SOHAIL KHOSHNEVIS 


Institute of Microbiology and genetics 

George-August University Goettingen 

Justus-von-Liebig-Weg 11 


Germany 

skhoshn@gwdg.de 

MICHAEL KIEBLER 

Medical University of Vienna 

Spitalgasse 4 

1090 Vienna 

Austria 

michael.kiebler@meduniwien.ac.at 

JEFFREY KIEFT 

Howard Hughes Medical Institute 

12801 East 17th Avenue 



jeffrey.kieft@ucdenver.edu 

JUDITH KIMBLE 

University of Wisconsin-Madison 

341E Biochemistry Addition 

433 Babcock Drive 

Madison, WI 53706-1544 


jekimble@wisc.edu



TERRI KINZY 

UMDNJ Robert Wood Johnson 

Medical School 

675 Hoes Lane 

08854 Piscataway, New Jersey 


kinzytg@umdnj.edu 

CHARLOTTE KNUDSEN 

University of Aarhus 

Gustav Wieds Vej 10C 

DK-8000C Århus 

Denmark 

crk@mb.au.dk 

JON HALVOR KNUTSEN 

Institute for Cancer Research 

0310 Oslo 

Norway 

jon.halvor.knutsen@rr-research.no 

REBECCA KOHLER 

ETH Zuerich 

Institute for Molecular Biology and 

Biophysics 

8093 Zuerich 


kohlerre@mol.biol.ethz.ch 

ANASTASSIA KOMAROVA 

Pasteur Institute 

Laboratoire de Génomique Virale et 

Vaccination, 28 rue du Dr. Roux 

75724 Paris Cedex 15 

France 

stasy@pasteur.fr 

338 

HARALD KÖNIG 

Forschungszentrum Karlsruhe GmbH 

Hermann von Helmholtz-Platz 1 

76344 Eggenstein-Leopoldshafen 

Germany 

harald.koenig@itg.fzk.de 

JOANNA KOWALSKA 


ul. Krakowskie Przedmiescie 26/28 

00-927 Warsaw 


asia@biogeo.uw.edu.pl 

GÜNTER KRAMER 

University of Heidelberg 

INF 282 


Germany 

g.kramer@zmbh.uni-heidelberg.de 

HEIKE KREBBER 

Philipps-Universität Marburg 

Emil-Mannkopff-Str. 2 

35037 Marburg 

Germany 

krebber@imt.uni-marburg.de 

POLINA KRJUCHKOVA 

Engelhardt Institute of molecular 

biology, RAS 

32, Vavilov str., Moscow, Russia 

119991 Moscow 


polina.krjuchkova@gmail.com

ANNA KROPIWNICKA 


Krakowskie Przedmiescie 26/28 

00-927 Warsaw 


akropiwn@tlen.pl 

DOROTA KUBACKA 



00-927 Warsaw 


dkuba@biogeo.uw.edu.pl 

ANDREAS KUHN 

Universitätsmedizin der Johannes 

Gutenberg-Universität Mainz 

Experimentelle und Translationale 

Onkologie 

55129 Mainz 

Germany 

kuhnan@uni-mainz.de 

L 

SVEN LAMMICH 

Ludwig Maximilians Universität 

München 

80336 München 

Germany 

slammich@med.uni-muenchen.de 

339 

MARKUS LANDTHALER 


BIMSB 

Max-Delbrück-Center for Molecular 

Medicine 

13125 Berlin 

Germany 

markus.landthaler@mdc-berlin.de 

MARLON LAWRENCE 

University of Maryland, Baltimore 

County 

UMBC 



law4@umbc.edu 

MICHAEL LEICHTER 

Université de Strasbourg, CNRS 

IBMC, 15 rue René Descartes 


France 

m.leichter@ibmc.u-strasbg.fr 

RACHEL LERNER 

University of North Carolina- Chapel 

Hill 

207 Fordham Hall 

27599 Chapel Hill, North Carolina 


rlerner@email.unc.edu



ALAIN LESCURE 

Strasbourg University - CNRS 


France 

a.lescure@ibmc.u-strasbg.fr 

KIN-MEI LEUNG 


Downing Street 

CB2 3DY Cambridge 


kml26@cam.ac.uk 

ACHIM LEUTZ 

Max-Delbrueck-Center for Molecular 

Medicine 

Robert-Roessle-Str. 10 

13125 Berlin 

Germany 

aleutz@mdc-berlin.de 

NOA LIBERMAN 

Weizmann Institute of science 

Rehovot 76100, PO Box 26 

76100 Rehovot 

Israel 

noa.liberman@weizmann.ac.il 

ZHAORU LIN 

Department of Pathology, University 

of Cambridge 

Division of Virology 



zl246@cam.ac.uk 

340 

JING-YI LIN 

Chang Gung University 

Department of Medical Biotechnology 

and Laboratory 

333 Tao-Yuan 

Taiwan 

jylin@mail.cgu.edu.tw 

CHIEN-LING LIN 

University of Massachusetts Medical 

School 

373 Plantation St., suite 204 

01605 Worcester, Massachusetts 


Chien-Ling.Lin@umassmed.edu 

HOWARD LIPSHITZ 

University of Toronto 

1 King's College Circle 

M5S1A8 Toronto 

Ontario 

Canada 

howard.lipshitz@utoronto.ca 

ETTA LIVNEH 

Ben Gurion University 

Faculty of Health Sciences 

84105 Beer Sheva 

Israel 

etta@bgu.ac.il

RICHARD LLOYD 

Baylor College of Medicine 

One Baylor Plaza 

77035 Houston 

Texas 


rlloyd@bcm.edu 

MARCELO LOPEZ-LASTRA 

Laboratorio de Virología Molecular, 

Facultad de Medicina, Pontificia 

Universidad Católica de Chile 

Marcoleta 391 

00000 Santiago 

Chile 

malopez@med.puc.cl 

FABRIZIO LORENI 

University of Rome Tor Vergata 

via Ricerca Scientifica 

00133 Roma 

Italy 

loreni@uniroma2.it 

JON LORSCH 

Johns Hopkins University School of 

Medicine 

21205 Baltimore 



jlorsch@jhmi.edu 

341 

REINHARD LUEHRMANN 


MPI fuer Biophysikalische Chemie 

Am Fassberg 11 


Germany 

reinhard.luehrmann@mpi-bpc.mpg.de 

EUGENIE LUFT 

University Clinic of Erlangen 

Wasserturmstr. 3-5 

91054 Erlangen 

Germany 

e.luft@yahoo.de 

MACIEJ LUKASZEWICZ 



00-927 Warsaw 


mlukas@biogeo.uw.edu.pl 

PETER LUKAVSKY 

MRC LMB 

Hills Road 

CB2 0QH Cambridge 


pjl@mrc-lmb.cam.ac.uk 

RADOSLAW LUKOSZEK 

University of Potsdam 

AG Molecular Biology, Haus 20 

D-14476 Golm 

Germany 

lukoszek@uni-potsdam.de



CHRISTINE LUTTERMANN 

Friedrich-Loeffler-Institut 

Paul-Ehrlich-Str. 28 

72076 Tübingen 

Germany 

christine.luttermann@fli.bund.de 

M 

NANDINI MANICKAM 

University of Maryland Baltimore 

County 

UMBC, 1000 Hilltop Circle 



nmanick1@umbc.edu 

CHRISTOPHE MARIS 

ETH Zurich 

Institute of Molecular Biology and 

Biophysics 

8093 Zurich 


cmaris@mol.biol.ethz.ch 

ALINE MARNEF 


Sanger Building 



am781@cam.ac.uk 

342 

FRANCK MARTIN 

CNRS 

IBMC 15 rue Rene Descartes 


France 

F.Martin@ibmc.u-strasbg.fr 

ENCARNA MARTINEZ-SALAS 

Centro de Biologia Molecular 

Nicolas Cabrera, 1 

28049 Madrid 

Spain 

emartinez@cbm.uam.es 

STEFANO MARZI 

IBMC Strasbourg 

15, Rue René Descartes 


France 

s.marzi@ibmc.u-strasbg.fr 

DENISA MATEUS 

Universidade de Aveiro / CESAM 

Campus Universitário de Santiago 

3810-193 Aveiro 

Portugal 

denisa.mateus@ua.pt 

HANSRUEDI MATHYS 

Friedrich Miescher Institute 


4058 Basel 


Hansruedi.Mathys@fmi.ch

KEN MATSUMOTO 

RIKEN 

2-1 Hirosawa 

351-0198 Saitama 

Japan 

matsumok@riken.jp 

FABIENNE MAUXION 

CGM-CNRS 

Avenue de la Terrasse 

91198 Gif-sur-Yvette 

France 

mauxion@cgm.cnrs-gif.fr 

ROBERT MC MAHON 

National Institute for Cellular 

Biotechnology 

9 Dublin 


robert.mcmahon@dcu.ie 

RAÚL MÉNDEZ 

Centre for Genomic Regulation 

(CRG) 

C/ Dr. Aiguader, 88 


Spain 

raul.mendez@crg.es 

WILLIAM MERRICK 

Case Western Reserve University 

School of Medicine 

44106-4935 Cleveland, Ohio 


wcm2@case.edu 

343 

CELINE MESTEL 

NYU 

550 1st Ave 

10016 New York 


celine.mestel@nyumc.org 

ODED MEYUHAS 


The Hebrew University-Hadassah 


POBox 12272 

91120 Jerusalem 

Israel 

meyuhas@cc.huji.ac.il 

REMACHA MIGUEL 

Universidad Autonoma de Madrid 

Centro de Biología Molecular 

28049 Madrid 

Spain 

miguel.remacha@uam.es 

KAREL MIKULIK 

Asad.Sci.Czech Republic, 

Inst.Microbiol 

Videnska 1083 

14220 Prague 4 


mikulik@biomed.cas.cz



POHL MILON 


Chemistry 

Fassberg Str. 11 


Germany 

pmilon@mpibpc.mpg.de 

ERIC MISKA 


The Henry Wellcome Building of 

Cancer and Developmental Biology, 

Tennis Court Rd 

CB2 1QN Cambridge 


eam29@cam.ac.uk 

SARAH MOHAMMAD-QURESHI 

The University of Manchester 

The Michael Smith Building 



s.mohammad-2@manchester.ac.uk 

MARTIN MOKREJŠ 

Charles University, Faculty of Science 

Vinicna 5 

12843 Prague 


mmokrejs@iresite.org 

344 

FRANCESCA MORETTI 

EMBL 



Germany 

moretti@embl.de 

OLIVER MÜHLEMANN 

University of Bern 

Baltzerstrasse 4 

3012 Bern 


oliver.muehlemann@izb.unibe.ch 

CHRISTINE MÜLLER 

Leibniz Institute for Age Research - 

Fritz Lipmann Institute 

Beutenbergstr. 11 

07745 Jena 

Germany 

cmueller@fli-leibniz.de 

JAMES MUNRO 


1300 York Avenue 



jbm2002@med.cornell.edu

VANDA MUNZAROVA 

Institute of Microbiology, AS CR, v.v.i. 

Laboratory of Regulation of Gene 

Expression 

142 20 Prague 


munzarova@biomed.cas.cz 

NICOLO' MUSNER 

San Raffaele Scientific Institute - 

DIBIT 


20132 Milan 

Italy 

musner.nicolo@hsr.it 

ASTRID MUSNIER 

Institut National de la Recherche 

Agronomique 

UMR 6175 - INRA - CNRS - 

Université de Tours 

37380 Nouzilly 

France 

astrid.musnier@tours.inra.fr 

N 

ISABEL NAARMANN 

Martin-Luther-University Halle- 

Wittenberg 


06120 Halle (Saale) 

Germany 

isabel.naarmann@biochemtech.unihalle.de 

345 

YOSHIKAZU NAKAMURA 


4-6-1 Shirokanedai 

108-8639 Tokyo 

Japan 

nak@ims.u-tokyo.ac.jp 

JAGPREET NANDA 


Johns Hopkins School of Medicine 

725 N Wolfe Street 



jnanda2@jhmi.edu 

SAWSAN NAPTHINE 

Cambridge University 

Tennis Court road 



sn@mole.bio.cam.ac.uk 

MARIE NAVEAU 

Ecole Polytechnique-CNRS 

Route de Saclay 

91128 Palaiseau Cedex 

France 

marienaveau@hotmail.com 

GABRIELE NEU-YILIK 

MMPU EMBL/University of 

Heidelberg 

Im Neuenheimer Feld 156 


Germany 

gabyneu-yilik@web.de



ANNA NIEDZWIECKA 

Institute of Physics, Polish Academy 

of Sciences 

32/46 Lotnikow Ave. 

02-668 Warsaw 


annan@ifpan.edu.pl 

KLAUS NIELSEN 

Aarhus University 

Gustav Wieds Vej 10c 

8210 Aarhus 

Denmark 

klahn@bioxray.au.dk 

MICHAEL NIEPMANN 

Justus-Liebig-University Giessen 

Friedrichstrasse 24 

35392 Giessen 

Germany 

michael.niepmann@biochemie.med.u 

ni-giessen.de 

ANDRZEJ NIERADKA 

Erasmus Medical Center 

Dr. Molewaterplein 50 

3015 GE Rotterdam 


a.nieradka@erasmusmc.nl 

346 

DIERK NIESSING 

Helmholtz Zentrum München 

c/o Gene Center LMU 

81377 Munich 

Germany 

niessing@helmholtz-muenchen.de 

EMILY NIKOLIC 





eicj2@cam.ac.uk 

O 

THEO OHLMANN 

INSERM-ENS de LYON 

46 Allee d'Italie 

69364 Lyon 

France 

tohlmann@ens-lyon.fr 

RENÉ OLSTHOORN 

Leiden University 




olsthoor@chem.leidenuniv.nl

DIRK OSTARECK 

University Hospital Aachen 

Res.Group Experimental Intensive 

Care Medicine 

52074 Aachen 

Germany 

dostareck@biochemtech.uni-halle.de 

ANTJE OSTARECK-LEDERER 

University Hospital Aachen 

Experimental Research Group of 

Intensive Care Merdicine 

52074 Aachen 

Germany 

aostareck@biochemtech.uni-halle.de 

MARTIN OTT 

University Kaiserslautern 

Erwin Schrödinger Str.13 

67663 Kaiserslautern 

Germany 

martin.ott@biologie.uni-kl.de 

P 

ALENA PALESKAVA 


Chemistry 


37077 Göttingen 

Germany 

Alena.Paleskava@mpibpc.mpg.de 

347 


GRAHAM PAVITT 

Dr. Graham Pavitt 

Faculty of Life Sciences 



graham.pavitt@manchester.ac.uk 

XOCHITL PEREZ-MARTINEZ 

Instituto de Fisiologia Celular, 

Universidad Nacional Autonoma de 

Mexico 

Circuito Exterior s/n. Ciudad 

Universitaria 

04510 Mexico City 

Mexico 

xperez@ifc.unam.mx 

JOHN PHAM 

Cell Press 

600 Technology Square 



jpham@cell.com 

NICOLA PHILLIPS 


Centre of Biomolecular Sciences 



paxnp6@nottingham.ac.uk



PHILIPPE PIERRE 

CNRS 

Parc Scientifique de luminy, Case 

906 

13288 Marseille, cedex 9 

France 

pierre@ciml.univ-mrs.fr 

JULIE PILOTTE 

The Scripps Research Institute 

10550 North Torrey Pines 

92109 La Jolla, California 


jpilotte@scripps.edu 

HUGO PINHEIRO 

IPATIMUP 

4200-465 Porto 

Portugal 

hpinheiro@ipatimup.pt 

TERRA PLANK 

University of Colorado Denver 

Anschutz Medical Campus 

12801 E 17th Avenue 



terra-dawn.plank@ucdenver.edu 

348 

VITALY POLUNOVSKY 

University of Minnesota 

420 Delaware Street S.E., MMC 276 



polun001@umn.edu 

MARTIN POSPISEK 

Charles University 

Vinicna 5 

12844 Prague 2 


martin@natur.cuni.cz 

MICHAEL POWELL 





mlp34@cam.ac.uk 

TUIJA POYRY 


80 Tennis Court Road 



taap2@mole.bio.cam.ac.uk 

ANNE-CATHERINE PRATS 

Inserm 

Inserm U858, I2MR 

31432 Toulouse 

France 

anne-catherine.prats@inserm.fr

THOMAS PREISS 

Victor Chang Cardiac Research 

Institute 

2010 Darlinghurst (Sydney) 

New South Wales 

Australia 

t.preiss@victorchang.edu.au 

MARTIN PRESTELE 

TU Kaiserslautern 

Erwin-Schroedinger-Str. 13 

67663 Kaiserslautern 

Rheinland-Pfalz 

Germany 

martin.prestele@biologie.uni-kl.de 

MAYA PRIZANT 

Tel Aviv University 

Ramat Aviv, Tel Aviv 

69978 Tel Aviv 

Israel 

mayapriz@post.tau.ac.il 

STEPHANE PYRONNET 

INSERM U858 

BP 84225 

31 432 Toulouse 

France 

stephane.pyronnet@inserm.fr 

349 

Q 

ALESSANDRO QUATTRONE 


Laboratory of Translational Genomics 

Centre for Integrative Biology 

University of Trento 

Via delle Regole 101 

38060 Trento 

Italy 

alessandro.quattrone@unitn.it 

R 

ANABELA RAMALHO 

National Institute of Health Dr. 

Ricardo Jorge 

Av. Padre Cruz 

1649-016 Lisboa 

Portugal 

anabela.ramalho@insa.min-saude.pt 

JAANUS REMME 

University of Tartu 

Riia 23, 

51010 Tartu 

Estonia 

jremme@ebc.ee 

ROBERT RHOADS 

LSU Health Sciences Center 

1501 Kings Highway 

71130-3932 Shreveport, Louisiana 


rrhoad@lsuhsc.edu



LUÍS RIBEIRO 

Center for Neuroscience and Cell 

Biology, University of Coimbra 

Largo Marquês de Pombal 

3004-517 Coimbra 

Portugal 

lfribeiro0@gmail.com 

EMILIANO RICCI 

Inserm U758 Ecole Normale 

Supérieure de Lyon 

46, Allée d'Italie 


France 

emiliano.ricci@ens-lyon.fr 

ADELE RICCIARDI 

University of North Carolina- Chapel 

Hill 

207 Fordham Hall 

27599 Chapel Hill, North Carolina 


adele@email.unc.edu 

JOEL RICHTER 

University of Massachusetts Medical 

School 

373 Plantation Street 

01605 Worcester, Massachusetts 


joel.richter@umassmed.edu 

350 

STAMATIS RIGAS 

Agricultural University of Athens 

Iera Odos 75 

118 55 Athens 

Greece 

srigas@aua.gr 

MARINA RODNINA 

MPI for Biophysical Chemistry 



Germany 

rodnina@mpibpc.mpg.de 

KOBI ROSENBLUM 

Haifa University 

Mount Carmel 

31905 Haifa 

Israel 

kobir@psy.haifa.ac.il 

VALENTINA RUGGERI 

San Raffaele Scientific Institute 


20132 Milano 

Italy 

ruggeri.valentina@hsr.it

DAVIDE RUGGERO 

University of California, San 

Francisco 

UCSF Mission Bay Campus 

94158 San Francisco 

California 


davide.ruggero@ucsf.edu 

LYUBOV RYABOVA 

Institut de Biologie Moléculaire des 

Plantes (IBMP), UPR CNRS 2357 

12, rue du Génèral Zimmer 


France 

lyuba.ryabova@ibmp-ulp.u-strasbg.fr 

INCHEOL RYU 

POSTECH 

Molecular Virology Laboratory 

PBC, Pohang University of Science 

and Technology 

San31, Hyoja-dong 

790-784 Pohang 

Republic of Korea 

grisip@postech.ac.kr 

S 

ADESH SAINI 

NICHD, NIH 

Building 6, Room 233 

20892-2716 Bethesda 



sainiade@mail.nih.gov 

351 

ARZU SANDIKCI 


ZMBH 

INF 282 


Germany 

a.sandikci@zmbh.uni-heidelberg.de 

BRUNO SARGUEIL 

CNRS 

Faculté de Pharmacie 

75270 Paris Cedex 

France 

bruno.sargueil@parisdescartes.fr 

EVELYN SATTLEGGER 

Massey University 

Private Bag 102904 

0632 Auckland 

New Zealand 

e.sattlegger@massey.ac.nz 

CHRISTIANE SCHAFFITZEL 

EMBL 

6 Rue Jules Horowitz 

38042 Grenoble 

France 

schaffitzel@embl.fr 

CLAUDIA SCHECKEL 

Friedrich Miescher Institute 


4056 Basel 


Claudia.Scheckel@fmi.ch



LUCA SCHENK 

ETH 

Wolfgang-Pauli-Str. 10 

CH-8093 zurich 


luca.schenk@pharma.ethz.ch 

GERT SCHEPER 

VU University Medical Center 

p/a De Boelelaan 1085 

1081HV Amsterdam 


gc.scheper@vumc.nl 

DIMITRI SCHERBAKOV 

Institute of Medical Microbiology, 

Zürich University 

Gloriastrasse 32 

8006 Zürich 


dscherbakov@imm.uzh.ch 

T. MARTIN SCHMEING 

LMB, Cambridge 

Hills Road 

CB2 OQH Cambridge 


schmeing@mrc-lmb.cam.ac.uk 

ROBERT SCHNEIDER 

NYU School of Medicine 

550 First Avenue 



robert.schneider@nyumc.org 

352 

EDUARD SCHREINER 

Beckman Institute 

405 N. Mathews 

61801 Urbana 

Illinois 


eschrein@ks.uiuc.edu 

MICHAL SHAPIRA 

Ben Gurion University of the Negev 

POB 653 

84105 Beer Sheva 

Israel 

shapiram@bgu.ac.il 

IVAN SHATSKY 

Moscow State University, Belozersky 

Institute of Physico-Chemical Biology 

Khokhlov str., Bldg. "A" 

119992 Moscow 


shatsky@genebee.msu.su 

MIKHAIL SHCHEPETILNIKOV 

IBMP 

12, rue du general Zimmer 


France 

mikhail.shchepetilnikov@ibmp-ulp.ustrasbg.fr

MICHAEL SHEETS 

Univ. of Wisconsin 

1300 University Avenue 

53706 Madison, Wisconsin 


mdsheets@wisc.edu 

NARA SHIN 

Yonsei Univ. College of Medicine 

new building 512(5th floor) Yonsei 

Univ. College of Medicine 

120-752 Seoul 

Republic of Korea 

grandciel@hanmail.net 

NIKOLAY SHIROKIKH 

Institute of Protein Research RAS 

4, Institutskaya st. 

142290 Pushchino 


nikolay.shirokikh@vega.protres.ru 

RACHEL SIMMONDS 

Imperial College 

Kennedy Institute of Rheumatology 

W6 8LH London 


r.simmonds@imperial.ac.uk 

ANGELITA SIMONETTI 

IGBMC Strasbourg 

1, rue Laurent Fries 


France 

simonett@igbmc.fr 

353 

MASAAKI SOKABE 


School of Medicine, University of 

California, Davis 

One Shields Ave, 327 Briggs (C/O 

Chris Fraser's lab) 

95616 Davis, California 


msokabe@ucdavis.edu 

NAHUM SONENBERG 


Rosalind and Morris Goodman 

Cancer Centre 


1160 Pine Avenue West 

Montreal, Quebec H3A 1A3 

Canada 

nahum.sonenberg@mcgill.ca 

JOERG SOPPA 

Goethe University, Frankfurt 

Biocentre 

D-60438 Frankfurt 

Germany 

soppa@bio.uni-frankfurt.de 

RICARDO SOTO RIFO 

Inserm U758 Ecole Normale 

Supérieure de Lyon 

46, Allée d'Italie 


France 

ricardo.soto-rifo@ens-lyon.fr



CHRISTIAN SPAHN 

Charite - Universitätsmedizin Berlin 

Ziegelstr. 5-9 

10117 Berlin 

Germany 

christian.spahn@charite.de 

KEITH SPRIGGS 





keith.spriggs@nottingham.ac.uk 

JEFF SQUIRES 

Victor Chang Cardiac Research 

Institute 

2010 Darlinghurst 

New South Wales 

Australia 

j.squires@victorchang.edu.au 

NANCY STANDART 



CB21GA Cambridge 


nms@mole.bio.cam.ac.uk 

354 

ABIGAIL STEVENSON 

Manchester Interdisciplinary 

Biocentre 

University of Manchester 

M1 7DN Manchester 


abigail.stevenson@manchester.ac.uk 

GEORG STOECKLIN 

German Cancer Research Center 


Germany 

g.stoecklin@dkfz.de 

NADINE STÖHR 

University of Halle 

Heinrich-Damerow-Str. 1 

06120 Halle 

Germany 

nadine.stoehr@medizin.uni-halle.de 

SURESH SUSMITHA 

University of Maryland, Baltimore 

County 

1000 Hilltop Circ 



susmi1@umbc.edu 

SYLWIA SZCZEPANIAK 

Department of Genetics, Warsaw 

University 

Pawinskiego 5A 

02-106 Warsaw 


sylwiaa.szczepaniak@gmail.com

DAN SZYMANSKI 

Purdue University 

1150 Lily Hall of Life Sciences 

47906 W. Lafayettte 

Indiana 


dszyman@purdue.edu 

T 

ABDESSAMAD TAHIRI-ALAOUI 

Institute for Animal Health 

Compton 

RG20 7NN Newbury 


abdou.tahiri-alaoui@bbsrc.ac.uk 

NONO TAKEUCHI 


5-1-5, Kashiwano-ha FSB401 

277-8562 Kashiwa 

Japan 

nono@k.u-tokyo.ac.jp 

JUDY TELLAM 

Queensland Institute of Medical 

Research 

300 Herston rd 

4006 Brisbane, Queensland 

Australia 

Judy.Tellam@qimr.edu.au 

355 


CLAUDIA TEMME 

University of Halle 


06120 Halle 

Germany 

ctemme@biochemtech.uni-halle.de 

ILYA TERENIN 


Leninskie gory 

119992 Moscow 


terenin@genebee.msu.ru 

ROLF THERMANN 




Germany 

rolf.thermann@embl.de 

CHRISTIAN THOMA 

University Hospital of Freiburg 

Hugstetterstr. 55 

79106 Freiburg 

Germany 

christian.thoma@uniklinik-freiburg.de 

SUNNIE THOMPSON 

University of Alabama at Birmingham 

BBRB 466/Box 21 

35294-2170 Birmingham 


sunnie@uab.edu



MARY THOMPSON 

Massachusetts Institute of 

Technology 

39 Clinton St., Apt. 5 

02139 Cambridge, MA 


marykayt@mit.edu 

JULIANO TOLEDO 

Universidade de São Paulo 

Av. Bandeirantes, 3900 

14025110 Ribeirão Preto - São Paulo 

Brazil 

jstoledo@gmail.com 

LEONARDO TRABUCO 

Center for Biophysics and 

Computational Biology and Beckman 

Institute 

University of Illinois at Urbana 

Champaign 

405 N Mathews Ave 

61801 Urbana, Illinois 


ltrabuco@gmail.com 

FELIX TRITSCHLER 

Max-Planck-Institute for 


Spemannstr.35 


Germany 

felix.tritschler@tuebingen.mpg.de 

356 

BECKY TSAI 

University of California, Irvine 

19182 Jamboree Blvd 

92697 Irvine, California 


kptsai@uci.edu 

JOSEPH TA-CHIEN TSENG 

National Cheng-Kung University 

No. 1 University Rd. 

701 Tainan 

Taiwan 

tctseng@mail.ncku.edu.tw 

TAMIR TULLER 

Tel-Aviv University 

School of Computer Science 

69978 Tel-Aviv 

Israel 

tamirtul@post.tau.ac.il 

NILGUN TUMER 

Rutgers University 

59 Dudley Road 

08901 New Brunswick 

New Jersey 


tumer@aesop.rutgers.edu

V 

LEOS VALASEK 

Institute of Microbiology, AS CR 

Videnska 1083 

14220 Prague 


valasekl@biomed.cas.cz 

LILY VARDIMON 

Tel Aviv University 


69978 Tel Aviv 

Israel 

vardi@post.tau.ac.il 

UMESH VARSHNEY 

Indian Institute of Science 

560012 Bangalore 

India 

uvarshney@gmail.com 

NORA VAZQUEZ-LASLOP 

University of Illinois at Chicago 

Cntr.Pharm.Biotech. - m/c 870 

60607 Chicago, Illinois 


nvazquez@uic.edu 

357 

BETHANY VEO 


University of Colorado Denver School 

of Medicine 

12801 E 17th Ave. 

80045 Aurora 

Colorado 


bethany.veo@ucdenver.edu 

GABRIELLA VIERO 

Center for Integrative Biology- 

University of Trento 

Via delle Regole, 101 

38100 Mattarello (Trento) 

Italy 

viero@science.unitn.it 

ANA VILLALBA 

Fundacio Privada Centre De 

Regulacio Genomica 

Dr. Aiguader, 88, 6th floor 


Spain 

ana.villalba@crg.es 

TALILA VOLK 

Weizmann Institute 

Department of Molecular Genetics 

76100 Rehovot 

Germany 

lgvolk@weizmann.ac.il



VIVIANA VOLTA 

San Raffaele del Monte Tabor 

Foundation 

via Olgettina 58 

20132 Milano 

Italy 

volta.viviana@hsr.it 

MARIEKE VON LINDERN 

Erasmus MC 

PO Box 2040 

3000 CA Rotterdam 


m.vonlindern@erasmusmc.nl 

REBECCA VOORHEES 

Medical Research Council: 

Laboratory of Molecular Biology 

Hills Road 

CB2 0QH Cambridge 


voorhees@mrc-lmb.cam.ac.uk 

VACLAV VOPALENSKY 

Charles University in Prague 

Vinicna 5 

12844 Prague 


vasek@natur.cuni.cz 

358 

W 

DOMINIQUE WEIL 

CNRS 

7 rue Guy Moquet 

94800 Villejuif 

France 

weil@vjf.cnrs.fr 

JOSEPH WETTSTEIN 

F. Hoffmann-La Roche Ltd 

Grenzacherstrasse 124 

4070 Basel 


joseph_g.wettstein@roche.com 

STEN WIE 

University of Colorado Denver 

RC1-South 

80045 Aurora 

Colorado 


sten.wie@ucdenver.edu 

HANS-JOACHIM WIEDEN 

University of Lethbridge 

4401 University Drive W 

T1K 3M4 Lethbridge 

Alberta 

Canada 

hj.wieden@uleth.ca

ANIA WILCZYNSKA 

Univerisity of Cambridge 




aw440@cam.ac.uk 

JIM WILHELM 

UC San Diego 

9500 Gilman Drive 

92093-0347 La Jolla 

California 


jwilhelm@ucsd.edu 

MARK WILLETT 

University of Sussex 

2C22, JMS Building 

BN1 9QG Brighton 


m.willett@sussex.ac.uk 

ANNE WILLIS 

Nottingham University 


NG72RD Nottingham 


anne.willis@nottingham.ac.uk 

WOLFGANG WINTERMEYER 

MPI for Biophysical Chemistry 



Germany 

wolfgang.wintermeyer@mpibpc.mpg.de 

359 

ANNIKA WOLF 


Martin-Luther-Universität Halle- 

Wittenberg 

Institute for Pathophysiology 

06114 Halle 

Germany 

annika.wolf@medizin.uni-halle.de 

DIETER WOLF 

Burnham Institute for Medical 

Research 

10901 North Torrey Pines Road 

92037 La Jolla, California 


dwolf@burnham.org 

CONNIE WAI HONG WOO 

Columbia University 

630 W. 168th St. 



ww2214@columbia.edu 

ANNA WYPIJEWSKA 

Department of Biophysics 

Institute of Experimental Physics 



00-927 Warsaw 


aw@biogeo.uw.edu.pl



X 

JIANXIN XIE 

Cell Signaling Technology Inc. 

3 Trask Lane 

01923 Danvers, Massachusetts 


jxie@cellsignal.com 

Y 

AKIKO YANAGIYA 


1160 ave Des Pins Ouest 

H3A 1A3 Montreal, Quebec 

Canada 

akiko.yanagiya@mcgill.ca 

EMILIO YÁNGÜEZ 

Centro Nacional de Biotecnología 

(CSIC) 

C/ Darwin 3 

28049 Madrid 

Spain 

eyanguez@cnb.csic.es 

LAURE YATIME 

MBI - Aarhus University 

Aarhus University 

8000 Aarhus 

Denmark 

lay@mb.au.dk 

360 

LUCY YOUNG 





paxly1@nottingham.ac.uk 

CHIEN-HUNG YU 

Leiden University 




yuch@chem.leidenuniv.nl 

MARAT YUSUPOV 

IGBMC 

BP 10142 

67404 illkirch 

France 

marat@igbmc.fr 

Z 

IZABELA ZABOROWSKA 

National Institute for Cellular 

Biotechnology 

9 Dublin 


izabela.zaborowska2@mail.dcu.ie 

GONG ZHANG 

Universität Potsdam 

Eislebenerstr. 7 

10789 Berlin 

Germany 

gong.zhang@uni-potsdam.de

YONGLONG ZHANG 

UMDNJ-Robert Wood Johnson 


675 Hoes Lane 

08854-8021 Piscataway, New Jersey 


yonglongzhang@yahoo.com 

SONJA ZILOW 

Ludwig-Maximilians-University 

80336 Munich 

Germany 

sonja.zilow@med.uni-muenchen.de 

JOANNA ZUBEREK 



00-927 Warsaw 


jzuberek@biogeo.uw.edu.pl 

361 

List of Participants

Useful Telefonnumbers 

Internal Numbers: 

Registration counter/ Operon Foyer 8509 

Course & <strong>Conference</strong> Office 7702 

Photolab/ Audiovisual staff 7703, 7774 

Switchboard 8100 

Security 7782 

Ambulance 19222 

(Airport) Transfers: 

ABS Airport Transfer 06205-29205-0 

ABS mobile Mr. Krieg 0151-161-353-92 

Express Drive 07223-80 83 89-0 

24h Hotline 0172-723 50 50 

York-Airport Service 06226-990135 

Bus company Moonlight Tours 0176-1003-8178 

7360892/3 

Hotels: 

Anlage 

Alt Heidelberg 9150 

Backmulde 53660 

Bayrischer Hof 872880 

Central 20641 

Crowne Plaza 9170 

Goldener Falke 14330 

ISG Hotel 6600 (internal) or 3861-0 

Kohler 970097 

Monpti 604560 

Perkeo 14130 

Vier Jahreszeiten 24164 

Convention and Visitors Bureau: 

Heidelberger Kongress und Tourismus GmbH, Ziegelhäuser Landstraße 3, D-69120 

Heidelberg 

Tel. +49 (0) 62 21-14 22-0, Fax +49 (0) 62 21-14 22 22 

E-Mail: info@cvb-heidelberg.de 

362

Notes


EMBL Heidelberg, 09-13 September 2009

Notes



Notes



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EMBO Conference on Protein Synthesis and Translational Control

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