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predictions from an array experiment in collaboration with Group 2 (Valcarcel) and showed that all SRp premRNAsundergo unproductive AS in SW480 with epithelial properties, but not in the same cells grown underconditions where they show mesenchymal phenotype. Group 24 (Soreq) is studying genome-wide alterations in ASin neurodegenerative disease as well as pathogenic states such as stress/PTSD. They used Exon Arrays to study ASchanges in nucleated blood cells of Parkinson’s patients undergoing deep brain stimulation. Group 1B(Neugebauer) has established stable P19 cell lines harboring inducible short hairpin RNA expression constructs,targeting SRp20 and SRp75. Robust protein knockdown has already been demonstrated with this system. RNAsamples will be prepared from cells after knockdown of either AS factor and used or probe preparation andhybridization to mouse splice junction arrays. The results will contribute to ongoing studies that aim to identify genetargets of SR protein family members. Group 15 (Cáceres) used the CLIP protocol to identify targets of bothSF2/ASF and hnRNPA1. Some SF2 tags were differentially associated with RNA in different cellularcompartments, and SF2 was shown to influence both splicing and subsequent polysome association of some RNAs.hnRNPA1 tags were mainly intronic and some were associated with known alternative splicing events, and sometargets were found to be regulated by hnRNPA1 under stress conditions. Group 10b (Tocchini-Valentini) hasdeveloped specific methodologies, for application of the original cis- and trans- Archaeaexpress technology to novelexperimental analysis of splicing variants of biomedically relevant membrane receptor genes, in mouse cell linesand other eukaryotic model systems. The plant groups 8, 14, 18 (Barta, Brown, Jarmolowski) expanded their RT-PCR panel to 300 AS events, focusing on genes encoding RNA-interacting proteins, transcription factors and genesinvolved in stress responses and signalling. Analysis of the 300 events identified many novel transcripts – around25% more than expected. The panel was used to analyse the effects of mutations in cap binding complex and NMDand of SR protein overexpression, and has stimulated collaborations with many other plant scientists. Groups 6, 23,26 (Ast, Smith, Auboeuf), analyzed the set of >200 AS évents that were found by junction array to be affected byknockdown of PTB/nPTB. They found that PTB-repressed exons were associated with enrichment of known PTBbindingmotifs within the exons and upstream introns, and that in vivo they tended to be spliced to highest levels inbrain and muscle. As a first attempt to model the extent to which deep sequencing technologies might be used forthe detection of splice variants Group 28 (Zavolan) implemented a simulation of the process by which sequencetags are obtained. As expected, the coverage of splice junctions with the relatively short tags produced by the Solexatechnology is considerably lower than the coverage of individual exons. Taken together with the low abundance ofsome isoforms this oindicates that quantification of relative abundance of splice forms will be challenging. Groups2 and 23 (Valcarcel, Smith 23), in collaboration with R. Guigo (CRG Barcelona) used Solexa sequencing tocompare control and PTB/nPTB knockdown HeLa cells, obtaining ~40M sequence reads under each condition.Using reads that map to the genome (ie excluding splice junctions) they were able to identify quantitative changes ina number of splicing events, some of which had previously been identified by junction array, but some of whichwere novel.WP9 Complexity of spliceosomal proteomesGroup 1a (Lührmann) affinity-purified individual, human spliceosomal complexes (i.e., A, B and C complexes)formed in HeLa splicing extract under identical physiological conditions (150 mM salt) and determined their proteincomposition by mass spectrometry (collaboration with H. Urlaub, member 1C). About 30 proteins are releasedfrom the B complex upon transition to C, while ~35 proteins are newly recruited during catalytic activation/step1 ofsplicing. The Lührmann lab (1A) developed a novel 2D electrophoretic procedure which has been applied for theseparation of proteins derived from purified A, B and C – spliceosomal complexes. Proteins were subsequentlymass-spec-ed and quantified by staining. The system also proved useful in monitoring the dynamics of proteinabundances during the transition of the spliceosomal complexes from one state to the other. The Lührmann Lab(1A) treated 25S [U4/U6.U5] tri-snRNP particles with m-Maleimidobenzoyl-N-hydroxy-sulfosuccinimide ester(Sulfo-MBS) and separated the proteins by 2D gel electrophoresis. The 2D map revealed additional spots derivedfrom putatively crosslinked tri-snRNP-specific proteins. In another feasibility study the Lührmann group (1A)investigated the protein-protein contacts in the purified human Prp19/CDC5L complex. Group 12c (C Branlant)improved the MS2 tagging of RNA by subjected the RNP complexes formed in nuclear extract to UV irradiation.The Beggs lab (9) used chemical cross-linking combined with mass spectrometry to identify specific amino-acids inPrp8p and Cwc21p that are in contact with one another. They also identified potential kinase target sites in Cwc21.The Beggs lab (9) found that a fragment of Brr2 can interact strongly with the 2nd step helicase Prp16p andinteracts weakly with the first step helicase Prp2p. The Branlant lab studied protein association with the SLS3stem-loop structures containing most of the regulatory elements acting on the HIV-1 A3 acceptor site.9
To identify protein-protein interactions occurring within the purified RNP complexes, Group 12c (C Branlant)used a tagged protein to look for the partners of protein hnRNP K within RNPs formed at the HIV-1 A7 regulatoryregion. Group 12c has developed with A van Dorsealler (Strasbourg) conditions for analysis of small RNPcomplexes by mass spectrometry in native conditions.WP10 Post-translational modification and dynamic regulationGroup 4 (Akusjärvi) is studying the role of the protein kinase PKA in pre-mRNA splicing. Groups 1A(Lührmann) and 1C (Urlaub) have performed a large scale phosproteome analysis of the different spliceosomalcomplexes derived from human cells. This study has gained insight into the dynamics of phosphorylation and dephosphorylationduring splicing. In this same topic, Group 3 (Stamm) has analyzed how the phosphatase PP1regulates the interaction of the splicing factors SF2/ASF, tra2-beta1 and SRp30c with RNA. Groups 15, 16 and 22(Caceres, Carmo-Fonseca and Lamond) have used FRET/FLIM microscopy to analyze interactions amongdifferent spliceosome components. The Lamond group has identified novel interactions between hnRNP M and twospliceosomal proteins, CDC5L and PLRG1 in human cells. Group 12D (Bertrand) is studying the binding of U1-specific proteins to the transcription site of the HIV-1 reporter RNA. Group 15 (Caceres) has used the CLIPprotocol (see WP8) to identify RNA targets of several splicing factors, including the SR protein SF2/ASF, hnRNPA1 and the exon junction component, Acinus. Group 12A (Tazi) has established an unsuspected tight couplingbetween the splicing factor B52 and DNA Topoisomerase I during Drosophila development. Group 10A(Biamonti) has continued the studies of the alternative splicing of the c-Ron proto-oncogene. This group hasfocused on the regulatory mechanisms that control the level of splicing factor SF2/ASF during epithelial tomesenchymal transition. Group 20b (Srebrow) has continued her study on the regulation of Rac1 pre-mRNAalternative splicing in a model of epithelial-mesenchymal transition induced by matrix metalloproteases. They haveidentified hnRNPA1 and hnRNP A2 as two regulatory proteins that regulate this alternative splicing event. Group26 (Auboeuf) has continued the study of how post-translational modifications of the p68 RNA helicase affects thefunction of this protein in transcription and in splicing.WP11 Function of splicing factor isoformsSeveral groups study the function of variants of splicing factors generated by alternative splicing of theirpre-mRNA precursors. These include components of the core splicing machinery as well as regulatoryfactors involved in splice site selection. Group 1A (Lührmann), group 16 (Carmo-Fonseca) and group21 (Krämer) have analyzed through proteomics and RNA expression analyses the extent of variationgenerated by alternative splicing in snRNP components, the 3’ splice site recognizing factor U2AF35 andthe branch point recognizing factor SF1, respectively. Functional analysis of some of these variants is onits way, with interesting insights obtained regarding the function of some of these variants in cell cyclecontrol. Groups 1B (Neugebauer), 2 (Valcárcel), 8, 14 and 18 (Barta, Brown and Jarmolowski), 23(Smith), 25 (Stévenin) and 26 (Tazi) have analyzed the generation and function of alternatively splicedvariants in regulators of 5’ and 3’ splice site utilization, including TIA-1/TIAR, PTB/nPTB, SC35,SRp20 and other proteins of the SR and hnRNP families in Drosophila, mammals and plants. Thesestudies have uncovered regulatory circuits (involving both self-regulation and cross-regulation betweenfactors) which determine the cellular levels of these activities, sometimes in a tissue-specific fashion.Group 2 (Valcárcel) has pro<strong>file</strong>d the generation of splicing factor variants in Drosophila sexdetermination, upon activation of signaling pathways in Drosophila cells, in mammalian cell tissues, inHodgkin lymphoma cell lines and, in collaboration with Groups 20 (Kornblihtt) and 10 (Biamonti),upon upon UV irradiation and under conditions of altered cell motility indiced by overexpression of theoncogenic SR protein SF2/ASF. Significant changes in alternative splicing isoform expression have beenidentified in these conditions, a significant fraction of which affect the structure of 3’ UTRs, suggestingdistinct susceptibility to regulation by miRNAs ad other proteins involved in cytoplasmic RNAmetabolism.10
Page 3 and 4: TABLE OF CONTENTSA. PERIODIC ACTIVI
Page 5 and 6: 1 PUBLISHABLE EXECUTIVE SUMMARY EUR
Page 7 and 8: Dr. Davide Gabellini(28) Swiss Inst
Page 9: WP7 Molecular characterization of s
Page 13 and 14: expressed in a particular tissue. T
Page 15 and 16: Duchenne muscular dystrophy (DMD).
Page 17 and 18: WP21 Reachout to the broader RNA co
Page 19 and 20: 4.1 TABLE OF MILESTONES JPA MONTH 2
Page 21 and 22: Numerous plasmids, constructs, extr
Page 23 and 24: collaboration between EURASNET and
Page 25 and 26: 5.1 WORK PACKAGE REPORTSWork Packag
Page 27 and 28: • Ongoing regular addition of new
Page 29 and 30: Work Package 4 The Alternative Spli
Page 31 and 32: Work Package 6 In silico approaches
Page 33 and 34: alternative splicing.162 Compare sp
Page 35 and 36: 33 Agreement on pre-mRNA substrates
Page 37 and 38: its numerous binding sites on both
Page 39 and 40: Work Package 8 Genome-wide Analyses
Page 41 and 42: 173 Further development of data ana
Page 43 and 44: Problems and explanations for delay
Page 45 and 46: eveal that there is a dramatic chan
Page 47 and 48: Work Package 10 Post-translational
Page 49 and 50: mutational analysis that led to the
Page 51 and 52: Isolation of an active step I splic
Page 53 and 54: FBP11 and 21 with these sequences.
Page 55 and 56: Similarly to what observed for SF2/
Page 57 and 58: prp45-113 mutant, which is compatib
Page 59 and 60: cell lines. In contrast in the pres
Page 61 and 62:
genes with roles in the development
Page 63 and 64:
Structural analyses of several RRMs
Page 65:
Work Package 17 Staff Exchange and
Page 69 and 70:
Barralle's lab, Krämer is acting a
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alternative splicing) and higher ed
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Student Symposium “Decision Makin
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variation and suitability for use w
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Following feedback from members we
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Invited seminars: In addition to me
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Publications 2008Author Title Publi
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Author Title Publication EURASNET P
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Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Minutes of the General Assembly dec
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241 Evaluate available careerdevelo
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156 The EURASNET memberStamm publis
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y coarse-grained foldingsimulation.
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overexpressor and mutant lines(mont
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machinery with particularrespect to
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alternative splicing, using theEDI
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contemporary findings withinthe EUR
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Page 112 and 113:
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
Licht K, Medenbach J, Lührmann R,K
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Page 122 and 123:
9 TABULAR OVERVIEW OF MAJOR COST IT
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total costs * 18.891,12 52778.60 41
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other (the rest) 48,57 0 56.539,43t
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travel 6.964,91overhead 3.081,07oth
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Participant 1A Type of expenditure
Page 132 and 133:
Participant 1B - Karla Neugebauera)
Page 134 and 135:
Participant 2 - Juan Valcarcela) Wo
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Participant 3 - Stefan Stamma) Work
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Participant 4 - Göran Akusjärvia)
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Participant 5A/B - Peer Bork/Rolf A
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Major cost items• ResearchPersonn
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Participant 7 - Francisco Barallea)
Page 146 and 147:
Francisco Baralle gave a talk to a
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alternative splicing on human disea
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J. Beggs has had frequent contact w
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Page 154 and 155:
Page 156 and 157:
Participant 12A - Jamal Tazia) Work
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Participant 12B - Bertrand Séraphi
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Participant 12C - Christiane Branla
Page 162 and 163:
Participant 12D - Edouard Bertranda
Page 164 and 165:
Participant 13 - Daniel Schümperli
Page 166 and 167:
Participant 14 - John W. S. Browna)
Page 168 and 169:
Participant 15 - Javier F. Cáceres
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• ResearchPersonnelName WP Person
Page 172 and 173:
MiscellaneousOligonucleotidesUse of
Page 174 and 175:
Major cost items• ResearchConsuma
Page 176 and 177:
Page 178 and 179:
Participant 21 - Angela Krämera) W
Page 180 and 181:
Participant 22 - Angus Lamonda) Wor
Page 182 and 183:
Participant 23 - Chris Smitha) Work
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ChemicalsEnzymesLaboratory supplies
Page 186 and 187:
Participant 26 - Didier Auboeufa) W
Page 188 and 189:
tool for the molecular analysis of
Page 190 and 191:
Participant 29 - Frédéric Allaina
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protein and instead possesses the d
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+ overhead 4.730= total eligible co
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Claudia Tamaro (PhD) has begun work
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ETHZIIMCBUPFSOTON-HGDTOTALJoint Pro
Page 200 and 201:
Direct eligible costs 53.240,48 0,0
Page 202 and 203:
Direct eligible costs 18.720,07 0,0
Page 204 and 205:
13 REPORT ON THE DISTRIBUTION OF TH
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Report on the Distribution of the C
Page 208 and 209:
For most work packages here follows
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pathways (eg, Reactome ).While PAND
Page 212 and 213:
The possible dependence of the effe
Page 214 and 215:
In some treatments or mutants, simi
Page 216 and 217:
Work Package 12: Mis-splicing and D
Page 218 and 219:
acid. Furthermore, using antibodies
Page 220 and 221:
At the 2007 annual meeting in Ile d
Page 222 and 223:
Partner 13 has extensively studied
Page 224 and 225:
inhibitors/modulators of RNA splici
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18 MONTH JPA: WORKPACKAGE TIMECOURS
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15.3 GRAPHICAL PRESENTATION OF WORK
Page 230 and 231:
15.4 TABLE OF MILESTONES (MONTH 37-
Page 232 and 233:
156 The EURASNET memberStamm publis
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194 Bertrand will analyze indetail
Page 236 and 237:
216 Aubeouf and Neugebauer willcoll
Page 238 and 239:
238 Establishing joint PhDcommittee
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283 A unified browser for allknown
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296 Group 12a (Branlant) willuse th
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315 Lamond will use a FLIM-FRET bas
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329 GROUP 7 (F. BARALLE)will contin
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333 GROUP 27 (GABELLINI)will:1) foc
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345 Schümperli’s group isplannin
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366 Quarterly network e-mail atmont
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2. Sharing Resources, Technology an
Page 256 and 257:
5. Ensuring DurabilityWorkpackage d
Page 258 and 259:
7. Molecular Characterization of Sp
Page 260 and 261:
8. Genome-wide Analyses of Splicing
Page 262 and 263:
9. Complexity of Spliceosomal Prote
Page 264 and 265:
202 Srebrow will investigate the ro
Page 266 and 267:
12. Mis-splicing and DiseaseWorkpac
Page 268 and 269:
13. Co-transcriptional Mechanisms o
Page 270 and 271:
14. Chemical Biology and Therapeuti
Page 272 and 273:
15. Development of Enabling Technol
Page 274 and 275:
269 IFMs in 20091. “Alternative a
Page 276 and 277:
18. Career DevelopmentWorkpackage d
Page 278 and 279:
20. SMEs and Technology TransferWor
Page 280 and 281:
21. Reachout to the Broader RNA Com
Page 282 and 283:
15.7 WORK PACKAGE LIST (MONTHS 37-5
Page 284 and 285:
ETHZIIMCBUPFSOTON-HGDTOTALJoint Pro
Page 286 and 287:
AMU 5 29 8 42UAAR 5 145 16 1665 29
Page 288 and 289:
Joint Programme of Activities RESEA
Page 290 and 291:
60 month period 18 month periodRequ
Page 292 and 293:
Integration1. Young Investigator Pr
Page 294 and 295:
UCAM-DBIOC 2350,000x1/3 € 50,000x
Page 296:
the management team. Audit costs ar
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