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acid. Furthermore, using antibodies that are specific for the different phosphorylated forms of RNA pol II CTDin ChIP assays, we found that the prp45-113 mutation caused a defect in the recruitment of serine 2-phosphorylated pol II to the yeast ASC1 gene. Together, these results are compatible with a defect in elongationrather than initiation of transcription. In a genome-wide screen we found synthetic genetic interactions betweenthe prp45-113 mutation and genes encoding several chromatin remodeling factors. In the remaining months,Beggs will measure the effects of the prp45-113 mutation on transcription elongation directly by pol IItranscription run-on assay (Birse et al, 1998) and/or by measuring the kinetics of induction of a P GAL -regulatedgene in vivo (Mason & Struhl, 2005). In parallel, Neugebauer’s group has detected changes in cotranscriptionalspliceosome assembly upon overexpression of the transcription elongation factor, TFIIS.Deliverable 102 examines how the polymerase controls the release of the nascent transcript. The group ofCarmo-Fonseca had previously shown that transcripts derived from human β-globin genes containing splicesite mutations are retained at the site of transcription. A key connection between transcription and mRNPbiogenesis is provided by the carboxyl-terminal domain (CTD) of the largest subunit of RNA Polymerase II(RNA Pol II LS), which binds several proteins essential for pre-mRNA processing. In order to investigate therole of the CTD in β-globin transcript release, we generated murine erythroleukemia (MEL) cell lines thatexpress α-amanitin resistant RNA Pol II LS with either full-length or truncated forms of the CTD. MEL cellsexpressing wild-type RNA Pol II LS maintained transcriptional activity in the presence of α-amanitin, indicatingthat the exogenous protein was functional. The mammalian CTD, which is essential for normal cotranscriptionalmaturation of mRNA precursors, comprises 52 heptad repeats. We show that a truncated CTDcontaining 31 repeats (heptads 1-23, 36-38 and 48-52) is sufficient to support transcription, splicing, cleavageand polyadenylation. Yet, the resulting mRNAs are mostly retained in the vicinity of the gene aftertranscriptional shutoff. The retained mRNAs maintain the ability to recruit components of the exon junctioncomplex and the nuclear exosome subunit Rrp6p, suggesting that binding of these proteins is not sufficient forRNA release. We propose that the missing heptads in the truncated CTD mutant are required for binding ofproteins implicated in a final co-transcriptional maturation of spliced and 3’ end cleaved and polyadenylatedmRNAs into export-competent ribonucleoprotein particles. <strong>EURASNET</strong> support is acknowledged in theresulting paper: Custódio, N., Vivo, M., Antoniou, M., Carmo-Fonseca, M. (2007) Splicing and cleavageindependent requirement of RNA polymerase II CTD for mRNA release from the transcription site. J Cell Biol179(2):199-207.Mechanisms of splicing factor recruitment to sites of transcription (Deliverables 140 & 103) has received amajor boost from the strategy of tagging splicing factors with GFP on bacterial artificial chromosomes (BACs)and then stably integrating the BACs into cell lines of choice. This system has the following advantages:• the tagged splicing factor is expressed under the control of its own promoter; in extensivepreliminary studies by Neugebauer’s lab, it was shown that protein expression levels in thestable cell lines are almost uniformly 1.5-fold of endogenous, as if the cells simply have anadditional allele.• the same tag can be used alternatively for imaging, for standard pull-downs or for ChIP. Thisis a significant advantage for ChIP, where background levels vary from antibody to antibody –here, the same antibody is used for every factor of interest.• Moreover, splicing factors that simply do not “ChIP” with antibodies against endogenousepitopes (presumably a problem of epitope access) are detectable using antibodies against thetag.• The BACs are portable, in that they can be transfected into any cell line, regardless of speciesor cell type. This is a significant saving in time and effort when developing new cotranscriptionalalternative splicing assays.Neugebauer has completed our survey of model genes, such as c-fos and, with Biamonti, SAT III. A manuscriptis in preparation. Moreover, a number of the new deliverables are collaborative applications of this advance toother questions regarding co-transcriptional spliceosome assembly and/or recruitment of splicing regulators toendogenous gene targets. Currently we have tagged: CBP80, SmB, U1 70K, U2AF65, Prp8, PTB, SRp20, SF2,SC35, 9G8, SRp40, SRp55, SRp75.217
TABLE: Relationship of transcriptional elongation to alternative splicing: compilation of treatments that alterelongation rate and affect alternative splicing outcome (Deliverable 142)FACTOR ORTREATMENT5,6-dichlorofuranosylbenzimidazoleriboside(DRB)FlavopiridolCamptothecinTSA(Trichostatin A)5 aza cytidineEWSVP16SWI/SNFCHEMICALNATURESmall organicmolecule.Nucleotideanalogue.Small organicmolecule.Small organicmolecule.Small organicmolecule.Small organicmolecule.Nucleotideanalogue.ProteinProteinProteinUV irradiation - -Slow mutant (C4)of RNApolymerase IIProtein -BASICMECHANISM OFACTIONInhibition of pTEFb(Cdk9 subunit), theprotein kinase thatphosphorylates thecarboxy terminaldomain (CTD) ofRNA polymerase IIInhibition of pTEFb(Cdk9 subunit)Inhibition of DNAtopoisomerase IInhibitor of proteinacetyl transferasesInhibitor of DNAmethyl transferasesHuman celltranscription factorHerpes virustranscription factorHuman celltranscription factor.Chromatinremodelling factor.CONSEQUENCESTRANSCRIPTIONInhibitselongationInhibitselongationONtranscriptionaltranscriptionalCreates raodblocks totranscriptional elongationFavorshistonehyperacetylation, subsequentchromatin opening andtranscriptional elongationInhibits DNA methylationand favors chromatinopening and transcriptionalelongationStimulates transcriptionalelongationStimulates transcriptionalelongationInteracts with Sam68 andcreates raodblocks totranscriptional elongation.Provokes RNA polymerase IIhyperphosphorylation andinhibition of transcriptionalelongationDisplayslowertranscriptional elongationrateEXAMPLES OFGENES WHOSEALTERNATIVESPLICING ISAFFECTED-Fibronectin- Bcl-X-Fibronectin- NCAM- Bcl-X- Cyclin D1- Fibronectin- NCAM- Fibronectin- NCAM- Cyclin D1- Fibronectin- CD44- Fibronectin- Bcl-X- Fibronectin- NCAMOther small molecules that inhibit transcriptional regulators and impact splicing:H-7 Small molecule/ Inhibition of Cdk7/ Inhibits Pol II Ser5 phosphorylation/ Cyclin D1Isoquinoline-5-sulfonic 2-methyl-1-piperazideH-8 Small molecule/ Inhibition of Cdk7/ Inhibits Pol II Ser5 phosphorylation/ Cyclin D1N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride218
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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)
Page 168 and 169: Participant 15 - Javier F. Cáceres
Page 170 and 171: • 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 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 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
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13. Co-transcriptional Mechanisms o
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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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