PDF file: EURASNET Annual Report 2008

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Partner 13 has extensively studied the U7 snRNP involved in histone RNA 3' end processing and its assembly,which is mediated by the SMN complex. For several years this partner has engineered modified U7 version(called U7 SmOPT) which no longer function in maturation of histone pre-mRNA but can carry antisenssequences. U7 SmOPT-derived RNA equipped with antisense sequences targeting specific splice sites should beideally suited to manipulate the splicing patterns of individual target genes. Moreover, being embedded intosnRNP particles, intracellularly expressed antisense sequences should be highly stable and resistant tonucleases. These vectors prove to be useful tools to induce exon skipping in several pathological modelsincluding β-globin gene in β-thalassimia, dystrophin gene in DMD, the cyclophilin A gene and the multiplyspliced HIV-1 transcripts. However, this approach has never been tested in the context of exon inclusion.Since it has been hypothesised that the SMN2 C>T transition in exon 7 abolishes an ESE, Partner 19 designedan innovative strategy aimed at reintroducing an exogenous SF2/ASF binding site at the SMN2 exon 7 3'-ss.They rely on a so-called bifunctional oligonucleotide carrying an ESE tail linked to an antisense sequencenecessary for specific targeting (TOE). Partner 13 adapted this principle by combining an antisense U7 snRNAwith an additional ESE sequence capable of binding SF2/ASF. This strategy proved very efficient resulting inan exon 7 inclusion level superior to 30% in transient transfection. When integrated into stable Hela-SMN-LucFcell lines through a lentiviral vector, this construct even allows for a long term correction reaching 50%inclusion.To test the efficacy of this novel modified U7, Partner 13 has introduced in his lab a very severe mouse modelfor SMA where symptoms are clearly reduced and survival of the animals is prolonged. Moreover Partner 13has made significant progress towards engineering inducible U7 snRNA cassettes. The plan for the next 18months will be to introduce modified U7 in the model mice and test the efficacy of SMN2 correction.Work Package 15: Development of Enabling TechnologiesSeveral new technologies were investigated to enable a more complete and detailed understanding of alternativesplicing. Some feasibility studies revealed limits of technologies (e.g., D2 bombardment, improvement ofprotein-RNA cross-linking, partner 1c). Alternative strategies will be explored (see future plans below).Important progress has been achieved in mass spectrometry analysis of RNA-protein cross-links (partner 1a).This strategy should be useful for many partners in the NOE. Similarly, constructs for stable expression oftagged mRNA species, for studies on alternative splicing and targeted, post-transcriptional RNA modificationare now available (partners 1b, 3 and 10b). Development of enabling technology was also pursued in manyunanticipated directions, due to new need in specific areas, this included:- establishing splicing-sensitive microarray designs and data analysis (partner 2)- improving TAP tag purification and developing new protein interaction assays (partner 12b)- developing FLIM-FRET techniques for analyzing the interactions of protein splicing factors in vivo.- implementing new proteomics approaches for analyzing protein interaction partners using quantitativemass spectrometry.Future plans/new JPA1) Highly accurate ESI FT MS of cross-linked species will be performed. The exact masses of the cross-linkedspecies, determined in this way, will serve as input information for the identification of the cross-linked peptidespecies: The exact mass and marker ions derived from the cross-linked peptide and RNA moiety will allow theidentification of cross-linked species by database search using an appropriate search engine. This informationwill help all EURASNET collaborator and groups outside the network to map protein-RNA cross-links.2) Enrichment strategies for cross-linked RNA-peptides will be used to identify cross-linked proteins/peptides incore 12S U2 snRNP, tri-snRNPs and spliceosomal B and C complexes. A similar enrichment strategy will beused to systematically investigate proteins with RRM domains complexes with various RNAs (collaborationbetween H. Urlaub (partner 1c) and F. Allain (partner 29). Such a systematic study will help (in the absence ofhighly resolved 3D protein–RNA structures) to elucidate the “code” of RNA interaction mediated by RRMdomains.3) Mass spectrometry-based identification and characterization of isoforms of spliceosomal proteins involved in(alternative) splicing: we plan to identify subsets of isoforms of proteins by multiple reaction monitoring221
experiments. We will also attempt to use quantitative mass spectrometry analysis to monitor changes inintracellular protein location, including factors involved in alternative splice choice.4) The use of massive sequencing for the analysis of alternative splicing event will be investigated by severalpartners.5) The use of efficient mutagenesis to identify protein interaction interfaces, a possible low resolution substitutefor D2 bombardment, will be investigated. We will also investigate the use of FLIM FRET to monitor proteininteraction in vivo.6) A system for high throughput analysis of alternative splicing exon through cloning in minigenes will be setup.7) The feasibility of using ultracentrifucation to monitor assembly of RNA binding protein on RNA will betested.Work Package 19: Public Understanding of RNA BiologyThis work package has a central role in the network as it coordinates the activities aimed to inform not only thegeneral public but also teachers and medical doctors. Until now a scientific officer was employed who oversawand organized the many activities. Many general activities like the support and update the PUS part of theEURASNET webpage as well as taking care of the teaching materials or writing press releases will becontinued.In addition, we plan to support science events organised by EURASNET members in a way that EURASNETactivities are represented optimally and members are encouraged to organize such events.EURASNET Brochure: The concept of the extended EURASNET Brochure is finished in English as well as inGerman and the layout is currently established. The extended EURASNET brochure will inform the publicabout the impact of alternative splicing on life in general. Target groups should be informed about the importantrole of RNA processing in the mechanisms of human disease as well as in the developmental programme ofhigher organisms. Furthermore, a detailed insight into research topics and fields of all the network participantsis given. Presented articles are richly illustrated with pictures, schemes and drawings for an easy understandingof the topic. Target groups to be reached were selected to act as “amplifiers”. These are medical doctors,teachers (especially biology teachers, intending to further instruct their pupils about the topic of alternativesplicing) and higher educated pupils by themselves. The extent of the brochure will be about 30 pages. In theyear 2009 the English and the German version will be published. Depending on the acceptance of this brochurewe intend to translate it into other languages like French and Spanish.EURASNET Newsletter: We will continue this successful activity and report on our activities, exciting newsfrom the Alternative splicing field biannually. The newsletter will be disseminated among researchers in theRNA field and selected target groups in particular in the medical field. This newsletter will comprise articlesdescribing the impact of alternative splicing on one specific issue. A short description of the work of oneresearch group as well as background information about their specific research topic will be given.Media training workshop and media writing contest: To educate the scientists of the EURASNET incommunicating science to the public, a media writing contest and a media workshop will be organized in theframework of the 2 nd International EURASNET Conference. These activities were extremely successful in thefirst meeting and had a very good response from the scientific community.Work Package 20: SMEs and Technology TransferWe will use the in vitro splicing assay format developed to carry out a large scale small molecule inhibitorscreen using the chemical compound library and robotic screening platform established at the University ofDundee by Professor Julie Frearson (www.drugdiscovery.dundee.ac.uk/). Any resulting chemical inhibitors ofsplicing will be characterised and made available to the EURASNET groups. Stable cell lines will beestablished in which the expression of differently coloured fluorescent proteins provide a reporter system forpre-mRNA splicing activity in vivo. These cells will be used as a secondary, in vivo screen for any smallmolecules that inhibit splicing in vitro and may also be used in a primary screen for in vivo222
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TABLE OF CONTENTSA. PERIODIC ACTIVI
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1 PUBLISHABLE EXECUTIVE SUMMARY EUR
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Dr. Davide Gabellini(28) Swiss Inst
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WP7 Molecular characterization of s
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To identify protein-protein interac
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expressed in a particular tissue. T
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Duchenne muscular dystrophy (DMD).
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WP21 Reachout to the broader RNA co
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4.1 TABLE OF MILESTONES JPA MONTH 2
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Numerous plasmids, constructs, extr
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collaboration between EURASNET and
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5.1 WORK PACKAGE REPORTSWork Packag
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• Ongoing regular addition of new
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Work Package 4 The Alternative Spli
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Work Package 6 In silico approaches
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alternative splicing.162 Compare sp
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33 Agreement on pre-mRNA substrates
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its numerous binding sites on both
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Work Package 8 Genome-wide Analyses
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173 Further development of data ana
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Problems and explanations for delay
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eveal that there is a dramatic chan
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Work Package 10 Post-translational
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mutational analysis that led to the
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Isolation of an active step I splic
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FBP11 and 21 with these sequences.
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Similarly to what observed for SF2/
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prp45-113 mutant, which is compatib
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cell lines. In contrast in the pres
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genes with roles in the development
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Structural analyses of several RRMs
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Work Package 17 Staff Exchange and
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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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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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Licht K, Medenbach J, Lührmann R,K
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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
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Participant 1B - Karla Neugebauera)
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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)
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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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Participant 12A - Jamal Tazia) Work
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Participant 12B - Bertrand Séraphi
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Participant 12C - Christiane Branla
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Participant 12D - Edouard Bertranda
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Participant 13 - Daniel Schümperli
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Participant 14 - John W. S. Browna)
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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: Major cost items• ResearchPersonn
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
Page 184 and 185: 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
Page 192 and 193: protein and instead possesses the d
Page 194 and 195: + overhead 4.730= total eligible co
Page 196 and 197: Claudia Tamaro (PhD) has begun work
Page 198 and 199: 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
Page 206 and 207: Report on the Distribution of the C
Page 208 and 209: For most work packages here follows
Page 210 and 211: 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 224 and 225: inhibitors/modulators of RNA splici
Page 226 and 227: 18 MONTH JPA: WORKPACKAGE TIMECOURS
Page 228 and 229: 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
Page 234 and 235: 194 Bertrand will analyze indetail
Page 236 and 237: 216 Aubeouf and Neugebauer willcoll
Page 238 and 239: 238 Establishing joint PhDcommittee
Page 240 and 241: 283 A unified browser for allknown
Page 242 and 243: 296 Group 12a (Branlant) willuse th
Page 244 and 245: 315 Lamond will use a FLIM-FRET bas
Page 246 and 247: 329 GROUP 7 (F. BARALLE)will contin
Page 248 and 249: 333 GROUP 27 (GABELLINI)will:1) foc
Page 250 and 251: 345 Schümperli’s group isplannin
Page 252 and 253: 366 Quarterly network e-mail atmont
Page 254 and 255: 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
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15. Development of Enabling Technol
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269 IFMs in 20091. “Alternative a
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18. Career DevelopmentWorkpackage d
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20. SMEs and Technology TransferWor
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21. Reachout to the Broader RNA Com
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15.7 WORK PACKAGE LIST (MONTHS 37-5
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ETHZIIMCBUPFSOTON-HGDTOTALJoint Pro
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AMU 5 29 8 42UAAR 5 145 16 1665 29
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Joint Programme of Activities RESEA
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60 month period 18 month periodRequ
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Integration1. Young Investigator Pr
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UCAM-DBIOC 2350,000x1/3 € 50,000x
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the management team. Audit costs ar
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