Identification des elements cis d'arn et developpement d'un gene rapporteur pour caracteriser les facteurs d'epissage qui controlent l'expression du facteur de transcription pro-apoptotique TAF6delta

Apoptosis is crucial to the development and homeostasis of eukaryotic organisms. It is regulated at different levels of gene expression. In response to intra- and extra-cellular stimuli, transcription factors regulate the expression of pro-survival and pro-apoptotic proteins. These transcription factors facilitate the formation of the RNA polymerase II pre-initiation complex (PIC) to activate transcription. The PIC is composed of RNA Polymerase II (Pol II) and the general Pol II transcriptional factors including the major core promoter recognition factor TFIID. TFIID is a multiprotein complex containing the TATA-Binding Protein (TBP) and 14 TBP Associated Factors (TAFs), including TAF6, which is the focus of my master's thesis. TAF6 possess 5 alternative splicing isoforms (alpha, beta, gamma, delta, epsilon). TAF6alpha and TAF6delta possess opposing biological activities. They are produced via the alternative splicing of exon2 TAF6. In contrast to the major alpha variant that possesses anti-apoptotic and oncogenic activities, the minor delta variant is pro-apoptotic. In contrast to TAF6alpha, TAF6delta doesn't dimerize with TAF9 in TFIID because it lacks the alternative exon2 that is excluded by an alternative splicing event.;We used microarray analysis to show that the impact on the transcriptome of the loss of the TAF6alpha is highly distinct from the impact caused by induction of pro-death TAF6delta isoform by antisense splice switching oligonucleotides. Furthermore, the depletion the major TAF6alpha splice variant results in the loss of cell viability. The importance of the induction of TAF6delta in programmed cell death, taken together with previous results showing that TAFdelta induces p53-independent apoptosis in numerous cancer cell types, prompted us to undertake a dissection of the cis-acting RNA elements that control splicing of the TAF6delta variant. We developed a minigene system to study cis-acting RNA elements that control the expression of TAF6delta. The minigene includes the genomic sequence of TAF6 (exon 2 to exon3) is driven by CMV promoter and it recapitulates the alternative splicing pattern of endogenous TAF6alpha and TAF6delta. We employed a mutational analysis to identify cis-acting elements of the TAF6 pre-mRNA. Our data have identified one enhancer splice element in the constitutive exon 2. Within intron 2, two polyC and polyG elements could regulate the alternative splicing of TAF6delta. These motifs represent potential binding sites for the RNA-binding proteins hnRNP K and H respectively. We therefore tested the effect of the overexpression of hnRNP K and H on TAF6 alternative splicing and found no effect on the expression of endogenous TAF6delta. Moreover, we report that a single nucleotide change that appears to perturb RNA secondary structure at the proximal splice site, completely reverses the splicing pattern. This mutation favors the choice of the distal splice site (weakened site) instead of the proximal splice site (TAF6alpha). Our data also suggest that a splice regulatory element that favors TAF6alpha expression is present in alternative exon 2.;To enable the identification the splicing factors influencing the choice of splicing site, we designed a new splicing reporter system. Our new vector contains the sequence of our minigene added a premature truncated codon in the alternative exon 2 and fused in frame to the EGFP protein. The combination of transient transfection assays with splice site switching oligonucleotides was used to validate this system as monitored by flow cytometry. In the future this system could be used to produce stable reporter cells lines to identify the splicing factors involved to the regulation of alternative splicing by screening cDNA or siRNA libraries.;Thus, my thesis presents the first identification of cis-acting RNA elements that control the splicing of the pro-apoptotic splicing factor TAF6delta as well as the development of a splice reporter system designed to enable the identification of splicing factors that regulate TAF6delta expression.;Keywords : TAF6delta, apoptosis, alternative splicing, cis-acting elements, splicing factors.