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194 Bertrand will analyze indetail the binding of U1-specific proteins to thetranscription site of the HIV-1reporter RNA by FRAP,including (i) interpretation ofthe FRAP curves with adiffusion-reaction model, (ii)analysis of the binding of U1proteins on reporter RNAcontaining mutation in theirsplice donor sequence (eitherstabilizing or destabilizing U1binding), (iii) analysing thebinding of mutant proteins,unable to bind U1 snRNP orother partners. He willdetermine the recruitment ofsplicing factors characteristicof a given spliceosomalcomplex (e.g. A, B, or C) onthe transcription site of wildtypeand mutant MINXreporter RNAs, and initiatingthe analysis of their bindingdynamic by FRAP.198 Carmo-Fonseca will performFRET analysis of splicingfactor interactions in thespliceosome. She will usestable cell lines expressing atandem array of transgenestagged with the MS2-bindingsequence. In these cells, cotranscriptionalspliceosomeassembly is visualized as asingle fluorescent focus.(month 42)199 Lührmann will develop amore detailed picture of themode of action of hPrp4 inthe splicing machinery withparticular respect to possibleinteraction partners.Lührmann and Urlaub willestablish the phosphoproteomof human and yeastsplicesomes. Lührmann willuse phosphor-peptide specificantibodies for in situlocalization of activespliceosomes (month 42)200 Stamm will determine howPP1 regulates the interactionof RNA with the splicingfactos SF2/ASF, tra2-beta1and SRp30c. (month 42)10 Cáceres O CO 4210 Cáceres O CO 4210 Cáceres O CO 4210 Cáceres O CO 42233
202 Srebrow will investigate therole of Akt as a novel SRprotein kinase. Thesubcellular localization of SRproteins will be evaluated byobservation of SR-GFPfusions in the presence ofsiRNA against AKT, andother established SR proteinkinases, such as SRPK orCLK. (month 42)204 Description of isoformdiversity and changes in corespliceosomal components indifferent tissues (month 42)205 Analysis of effects of SF1isoform overexpression onalternative splicing eventsidentified by CLIP (month42)206 Investigate the role ofU2AF35 isoforms andU2AF35-related proteins onsplicing regulation / cell cycleprogression (month 42)208 Analyze SR protein andPTBL over-expression andmutant lines using the 384 ASRT-PCR panel (month 42)211 In depth characterization oftrans-acting factors ofparticular interest withregards to their functionality,biochemical properties, andgeneral importance for thecellular metabolismparticularly in thedevelopment of tumors(Biamonti) (month 42)215 To determine the molecularbasis for the effect of Prp45pon transcription, Beggs willanalyse the effects of prp45mutant alleles on therecruitment of CTD kinasesand chromatin remodelingfactors to the ASC1 gene byin vivo ChIP assay, and theinteraction of these proteinswith Prp45p will be tested inpull-down assays in vitro.(month 42)10 Cáceres O CO 4211 Valcárcel O CO 4211 Valcárcel O CO 4211 Valcárcel O CO 4211 Valcárcel O CO 4212 Baralle O CO 4213 Neugebauer O CO 42234
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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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Page 118 and 119:
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
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MiscellaneousOligonucleotidesUse of
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Major cost items• ResearchConsuma
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Participant 21 - Angela Krämera) W
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Participant 22 - Angus Lamonda) Wor
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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 222 and 223: Partner 13 has extensively studied
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 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
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
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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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