PDF file: EURASNET Annual Report 2008

Work Package 13 Co-transcriptional Mechanisms of AlternativeSplicingLead Participant: Karla Neugebauerdeliverables: 215 To determine the molecular basis for the effect of Prp45pon transcription, Beggs will analyse the effects of prp45mutant alleles on the recruitment of CTD kinases andchromatin remodeling factors to the ASC1 gene by in vivoChIP assay, and the interaction of these proteins withPrp45p will be tested in pull-down assays in vitro. (month42)216 Aubeouf and Neugebauer will collaborate to establishsplicing factor ChIP in MCF7 cell lines. These breastcancer cells are responsive to estradiol, which stimulatestranscription of cyclin D1, PS2 and other genes. Thiscellular model will allow us to test the splicing process ongene products made in response to hormones. We willestablish stable cell lines with integrated copies of BACsexpressing GFP-tagged versions of U1 70K, U2AF65,Prp8, CBP80 and members of the SR protein family, inorder to investigate the interplay between transcription, cotranscriptionalspliceosome assembly and AS outcome.(month 42)217 Kornblihtt will determine whether intronic siRNAs affectalternative splicing, using the EDI minigene reporter. If so,experiments will be initiated to determine whether theeffect is via a gene silencing mechanism involving histonemodifications. (month 42)218 Smith and Neugebauer will collaborate to determinewhether PTB is recruited co-transcriptionally to targetgenes identified in Smith lab by microarray analysis (seeWP8). PTB-GFP BACs will be established (if possible) andstably transfected into cell lines, including mouse C2C12myoblasts. Alternatively, PTB antibodies will employed insplicing factor ChIP. (month 42)219 Bertrand and Neugebauer will collaborate to determinewhether splicing of HIV nascent transcripts is cotranscriptional,using a model gene comprising a split MS2reporter. ChIP of the MS2 binding protein will indicatewhen and where along the gene splicing catalysis occurs.(month 42)monthplanned achieved42 on target42 on target42 3642 delayed(see newdeliverable)42 on targetObjectives of the work package• Analysis of the coupling between transcriptional and splicing regulation in order to ultimately explain how alternativemRNAs are expressed in vivo. The overall aim is to optimize and extend key techniques. A particular focus of this periodwill be exchanging expertise and reagents, such as Pol II mutants, antibodies, and newly established cell lines, forapplication among the experimental systems.Short description of activities215 Molecular basis for the effect of Prp45p on transcription.We have developed a faster sampling protocol for yeast RNA analysis and find that the kinetics of the ts defect of the prp45mutations are much faster than previously thought, with effects seen within 5 min of the temperature shift, and reaching amaximum by 20 to 30 min. We have therefore repeated and extended the analyses of the effect of prp45 mutations ontranscription. Using antibodies that are specific for the different phosphorylated forms of RNA pol II CTD in ChIP assays,we previously showed that the prp45-113 mutation causes a defect in the recruitment of serine 2-phosphorylated pol II to theyeast ASC1 gene. We have now demonstrated a defect in the recruitment of the Ser-2 kinases Bur1/Bur2 and Ctk1 in the55