PDF file: EURASNET Annual Report 2008

spliceosome. They have now focused on generating phosphor-specific antibodies in order to visualize catalytically activespliceosomes in the cell nucleus. For example, SF3b155 is a U2 snRNP specific protein that is specifically phosphorylatedonly upon spliceosome activation and its dephosphorylation is required for the second step of splicing. PhosphorylatedSF3b155 is thus specific for active spliceosomes. To visualize these by immunofluorescence (IF) in HeLa cells, theygenerated phospho-peptide specific antibodies (R79) specifically recognizing SF3b155 phosphorylation sites, which werefirst determined by mass spectrometry (collaboration with H. Urlaub). The antibodies were highly specific forphosphorylated SF3b155 on Western blots. IF studies with HeLa cells revealed a punctated staining pattern exclusively inthe nucleoplasm. Extensive further double IF studies revealed that the R79 signal i) was strictly dependent on ongoingtranscription, ii) did not overlap with nuclear speckles, and thus iii) constituted only a subset of all U2 snRNP containingregions within the nucleoplasm. Interestingly, the R79 signal co-localized with sites of newly synthesized RNA, consistentwith the idea of co-transcriptional splicing, but also at non-overlapping sites. Consistent with this observation, the R79 signalpartially overlapped with actively transcribing RNA polymerase II. Taken together, the data suggest a unique localization ofcatalytically active spliceosomes in the cell nucleus.200 PP1 regulation of the interaction of RNA with the splicing factos SF2/ASF, tra2-beta1 and SRp30c.Group 3 (Stamm) has previously shown that the phosphatase PP1 binds to the RRM of several splicing factors and regulatesalternative pre-mRNA processing. In the period of this report, this group showed that PP1 has two functions when binding tothe RRM of splicing factors. First, it dephosphorylates splicing factors as previously reported. The second function that isnow under investigation is that PP1 causes the release of RNA from the RRM.201 Relationship between B52 and the other candidate genes.A genetic screen performed by Group 12a (Tazi) resulted in the identification of DNA topoisomerase I (Topo I) as asuppressor of the developmental defects caused by overexpression of the Drosophila SR protein B52 in vivo. Thus, thisestablished an unsuspected tight coupling between the splicing factor B52 and Topo I during Drosophila development. Thisgroup now found that the localization of Topo I perfectly matches the localization of B52 on polytene chromosome at allloci. In transgenic flies, expression of a high affinity binding site for B52 restricted localization of both B52 and Topo I tothis single transcription site, whereas B52 RNAi knockdown induced mis-localization of Topo I in the nucleolus (Juge et al.,submitted). Unexpectedly, specific overexpression of B52 in salivary glands allowed massive recruitment of both B52 andTopo I, but not RNA Pol II, on nuclear RNA molecules, demonstrating for the first time that Topo I may assemble on RNPsto coordinate the action of SR proteins during transcription. This coordination likely involves an intrinsic kinase activity ofTopo I, which was shown to be required to specifically phosphorylate B52 in vivo. Thus, the partnership between Topo I andB52 for the specific phosphorylation of serine residues located within the RS domain and the control of DNA supercoïlingcould be key determinants for functional integration of transcription and RNA processing machineries in a mutuallybeneficial manner for efficient and regulated gene expression.202 Role of Akt as a novel SR protein kinase.Finally, Group 20b (Srebrow), a member YIP program, has continued her study on the regulation of Rac1 pre-mRNAalternative splicing in a model of epithelial-mesenchymal transition induced by matrix metalloproteases, in particular MMP-3 (stromelysin-1). They had already identified hnRNPA1 and hnRNP A2 as two regulatory proteins that were able to bindRac1 alternative exon 3b. They constructed an exon 3b splicing reporter minigene and used a set of biochemical assays suchas RNA-affinity purification, UV-cross linking followed by immunoprecipitation, and RNA gel shift, to start deciphering themechanism by which MMP-3 causes an increase in exon 3b inclusion and to further characterize the involvement of hnRNPA1. They defined a sequence element within exon 3b that resembles the hnRNP A1 consensus binding site. Mutation of thiselement results in a decreased binding of hnRNP A1 to this exon compared to the wild-type one. Furthermore, overexpressionof hnRNPA1 inhibits 3b exon inclusion in transcripts derived from the Rac1 3b minigene. Interestingly, theyfound that there is less hnRNP A1-exon 3b complex formation with nuclear extracts from MMP-3 treated cells than withnuclear extracts from untreated ones. These and other results from their lab suggest that other regulatory factors may becooperating with hnRNP A1 to repress inclusion of 3b exon under basal conditions and to induce its inclusion upontreatment with MMP-3. While exploring post-translational modifications of hnRNP A1 that could be involved in regulatingits activity upon MMP-3 treatment, they started working in the SUMOylation pathway and obtained unexpected resultssuggesting that certain SR proteins may influence this process.Problems and explanations for delays, postponements etc.202 Srebrow has encountered technical difficulties related to the growth and responsiveness of the cell culture system use tostudy Rac1 splicing regulation and they have spent considerable time and effort to overcome these problems. This hasresulted in lack of progress with respect to deliverable 202, the role of Akt as an SR protein kinase. This is now presented asa deliverable for the next period.PUBLICATIONSAkusjarvi,G. (2008) Temporal regulation of adenovirus major late alternative RNA splicing. Front. Biosci., 13, 5006-5015.Bessonov,S., Anokhina,M., Will,C.L., Urlaub,H. and Lührmann,R. (2008)49