Análisis de regiones específicas de la RNA Polimerasa II de sacharomyces cerevisiaeIdentificación y caracterización de moduladores transcripcionales que interaccionan con las mismas

Abstract

In eukaryotes, like saccharomyces cerevisiae, the enzyme needed for the synthesis of the mrna is the rna polymerase ii. The catalytic core of the bacterial and eukaryotic enzymes is highly conserved through evolution. Curiously enough, only five of the twelve subunits of rna pol ii have bacterial homologues, whereas that other six (rpb4, rpb5, rpb7, rpb9, rpb10 and rpb12) are common to archaea, but without eubacterial homologues. It has been established that the three eukaryotic rna polymerases have a conserved structure made of eleven subunits; Five of them correspond to bacterial enzyme and six are conserved with archaea. The twelfth, rpb8, is only eukaryotic. Structural data obtained during the last few years on s. Cerevisiae rna pol ii have allowed to draw a detailed map of the physical interactions between the different subunits, and to establish which regions are important for transcription. The knowledge of the architecture of this huge complex as well as the function of its different parts in transcription are key points to understand the role of each element in the different steps of transcription: From initiation to mrna export. Specific regions of the rna polymerase ii could be involved in the contact with transcriptional modulators. This contact can be direct or through the interaction with other general transcription factors (gfts) or specific factors. Based on these data, the main objective of our project is the analysis of specific regions of the rna pol ii that, according to its location at the surface of the complex, are candidates to contact transcriptional modulators, and to identify and analyse these putative transcriptional modulators. Analysis and identification of rna pol ii specific regions. Using bioinformatics tools (blast, multialin) we have carried out an extensive analysis searching for rna pol ii specific regions. Based on crystallographic data we have identified five rna polymerase ii specific regions localised on the surface of the complex structure, corresponding to the superior jaw, funnel, dock, foot and wall (figure r.2., table r.2.). These specific regions, due to their localisation have been selected as putative domains implicated in the contact with transcriptional modulators and then, probably involved in specific roles during different steps of transcription. In order to make this study we have selected those regions according to the existence of mutants described in the bibliography (table r.3.) with a clear phenotype of temperature sensitivity and some elongation drugs sensitivity. We have carried out this study with the foot and the upper jaw regions (rpb1) and the wall (region of rpb2). Identification of transcriptional modulators interacting to different rna pol ii specific regions looking for proteins that interact to these rna pol ii specific regions, we have carried out two-hybrid screenings with a s. Cerevisiae genomic library. In the case of the wall and the upper jaw we didn find any putative interacting protein. On the contrary, we identified three different proteins that interact with the foot region, corresponding to mvp1, rep1 and spo14 (figure r.12. And table r.6.). All of these proteins participate in different processes unrelated with the transcription. However, recent data propose a relationship between mvp1 and rep1 with mediator (hazbun et al., 2003; Hallberg et al., 2004), and show associations of mvp1 and spo14 with proteins involved in processes of mrna modification, mrna export and transcription regulation (vollert and uetz, 2004; Titz et al., 2006; Fromont-racine et al., 2000; Hairfield et al., 2001). Characterization of transcriptional modulators in order to corroborate the physical interaction identified by two hybrid analysis, we have carried out co-inmunopurification and co-inmunoprecipitation experiments. Using tagged mvp1 and spo14 proteins, with the c-lytag tag (biomedal, seville, spain) (figures r.16. And r.17.), we have demonstrated the physical interaction between mvp1 and rpb1 (figures r.19. And r.20.), as well as between spo14 and rpb1 (figure r.20.). Using the tagged mvp1 and spo14 in immunolocalisation experiments we have demonstrated that these proteins are in the nucleus, in addition to their well known cytoplasmic localisation (ekena and stevens, 1995; Huh et al., 2003) (figures r.21. And r.22.). To check for the association between mvp1 and/or spo14 with dna in vivo, we have carried out chromatin inmunoprecipitation (chrip-chip) analysis, using chips containing the orf of the s. Cerevisiae genes transcribed by the rna pol ii, as well as non coding regions, rdnas (18s, 25s) and rna pol iii (trna and scr1) genes (alberola et al., 2004). The results, using tagged mvp1 and spo14, clearly confirm that mvp1 protein bind dna fragments containing the promoter region of some genes. Contrary, spo14 associates in vivo with dna inside the orfs probably interacting with the rna pol ii during elongation, and we can not discard the association with promoter regions (figures r.26 to r.29). These proteins do not bind genes transcribed by rna pol i and iii, and neither non coding regions. The analysis of our chrip-chip data shows a slight positive correlation with transcription rates, when we compare with data from other authors (figures r. 32. And r.33.) (vicent pelechano and josé e. Pérez-ortín personal communication); (silvia jimeno doctoral thesis). In order to analyse and clarify more in detail the possible role of these proteins in transcription we have carried out, in vivo transcription assay measurements (gene length-dependent accumulation of mrna, glam (morillo-huesca et al., 2006)), by using mvp1 and spo14 mutants (euroscarf). The results show that none of these mutants affect transcription elongation (figure r.23). In addition, mvp1 and spo14 deletions do not significantly alter the mrna accumulation of some of their target genes, neither the accumulation of control act1 gene, when measured by real time rt-pcr (figures r.30. And r.31.). Based on the previously described genetic and physical interactions between vps1 and mvp1 (ekena and stevens, 1995), we have also analysed by glam the involvement of vps1 in transcription elongation. As it is the case for mvp1, the vps1 deletion (euroscarf), does not affect transcription elongation (figure r.23.). Genetic analysis between mvp1, vps1, spo14 and different mutants of transcriptional machinery components point to a genetic interaction between mvp1 and the rpo21-4 mutant (corresponding to the foot of the rna pol ii) (archambault et al., 1990), as well as between vps1 and the rpo21-4 mutant. However, we have not found any genetic interaction between spo14 and rpb1 mutants (figures r.35., r.36. And r.40. To r. 47.; Summary of the interactions in table r.9. ). All these data sign to the fact that mvp1 is a transcriptional modulator that has a role in association with the rna pol ii. However, we have not been able to define a function for mvp1 in the transcriptional process. Based on the interaction data between mvp1 and the yra2, std1 and srb7 proteins (vollert and uetz, 2004; Hazbun et al., 2003) and the putative relationship between vps1 and ceg1 (ekena and stevens, 1995; Gavin et al., 2002), we can not discard a possible role for mvp1 in mrna regulation, modification and/or export. Recent publications show a putative connection between spo14 and transcription. It has been demonstrated the interaction of spo14 with the decapping protein dpc2 (fromont-racine et al., 2000). There is also a relationship between spo14 and the ste12 transcription factor (hairfield et al., 2001). Spo14 is a member of phospholipase d (pld) family which synthesizes phospatidic acid, that, in term, stimulates kinases in several biological systems (jenkins and frohman, 2005; Hairfield et al., 2001). The ctd domain of the rna pol ii is phosphorilated, as well as the rpb2 and rpb6 subunits (kolodziej et al., 1990). Although we have not been able to stablish a role for spo14 in transcription, we could speculate that this protein could be implicated in phosphorylation processes of the rna pol ii during transcription. Functional characterisation of the foot rna pol ii specific region it has been described that the temperature sensitivity of rpo21-4 mutant (foot of the rna pol ii) is suppressed by the overexpression of rpo26, a subunit shared by the three eukaryotic rna polymerases (nouraini et al., 1997). In addition, rpb6 interacts in vivo with rpb8, another common subunit to all three eukaryotic rna polymerases, and the only typically eukaryotic (briand et al., 2001). Based on these data, we analysed if rpo21-4 phenotype could be suppressed by the rpb8 overexpression, and demonstrated that it is not case (figure r.48.). Structural data analysis shows that there is a contact between rpb5 and rpb6 and between these common subunits and the foot rna pol ii specific region. We also demonstrated that rpb5 overexpression do not suppress the rpo21-4 temperature sensitive phenotype. One possibility could be that the rpo21-4 mutation causes a conformational change in the foot region big enough to block the suitable interaction with rpb5 or that rna pol ii activity is mainly affected, in adition to the conformational alteration of the rna pol ii . Rpo21-4 in vivo transcription analysis, by glam, shows a defect in transcriptional elongation with no correlation with chromatin related processes (figures r.51. And r.53.). In order to investigate more in detail and to clarify the role of the foot rna pol ii specific region we carried out global expression analysis by macroarray hybridization (including the whole set of the s. Cerevisiae orfs) with rna from rpo21-4 and wild-type w303 strains. However, t-profiler analysis suggested chromosomes v and xvi duplications for the rpo21-4 mutant strain (figure r.57.). We have corroborated these aneuploidy by real time pcr with genes of the different arms of these chromosomes (figures r.62, r.63 and r.65.). To rule out that the macroarray data are only the result of the chromosomes duplication we represented the highest values obtained in these analysis over the different chromosomes (figure r.66.) showing that genes with highest values of expression are dispersed over all the chromosomes. Despite the fact that a preliminary analysis of the macroarray experiments suggests a relationship between rpo21-4 gene expression and saga machinery (tata-less promoter), we can not exclude an indirect effect due to chromosomes v and xvi duplications. We also propose that repression by nc2 and mot1 (and/or probably others) is affected in the rpo21-4 mutant (model, figure d.1.) new mutants of the specific region of the foot we have generated a new mutant of this specific region of the rna pol ii. Mutations, by aminoacid substitutions (i986v and l1026a), rend a temperature sensitivity phenotype. However, this mutant is 6-azauracile and micophenolic acid resistant. The fact that these residues are not exposed (located inside the rna pol ii complex) could explain the fact that there is no genetic interaction neither with mvp1 nor vps1, at a difference than this occurred with rpo21-4 mutant. Functional analysis of the upper jaw rna poll specific region the three different mutants rpo21-18, rpo21-24 (archambault et al., 1992) and rpb1-19 (scafe et al., 1990) corresponding to the jaw rna pol ii specific region show temperature sensitivity phenotypes. In addition, rpo21-18 and rpo21-24 are 6-azauracile and micophenolic acid sensitive. It has been described a genetic and physical interaction between rpo21-18 and rpo21-24 mutants and the tfiis transcription factor. In fact, the corresponding gene (dst1 or ppr2) overexpression suppressed the rpo21-18 and rpo21-24 6-azaurazile sensitive phenotypes (wu et al., 1996). In addition, in vivo and in vitro results show that the jaw region interacts with tfiis (malagón et al., 2004). In the attempt to characterise more in detail the putative role of the jaw rna pol ii specific region, we analysed if overexpression of dst1 also suppressed the mutant phenotypes. As we show in figures r. 73. And r.74., the rpb1-19 and rpo21-18 mutant temperature sensitivity phenotype are also suppressed by tfiis overexpression. In addition, in vivo transcription analysis, by glam, shows that the three mutants are affected in transcriptional elongation (figure r.75.). Our results, by glam analysis, also demonstrated that tfiis overexpression corrects the transcriptional elongation defect of rpo21-18 and rpo21-24 mutant strains (and probably it is also the case for the rpb1-19 mutant) (figure r.76.). Our data and these of other authors point to a role of the jaw rna pol ii specific region in transcription elongation in association with tfiis transcription factor.