Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x S splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.

Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-x s ) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-x S 5= splice site.T he expression of protein-coding genes in eukaryotes is a highly orchestrated process that involves multiple coordinated events. Genomic DNA must be transcribed into precursor mRNAs (pre-mRNA) by RNA polymerase II (RNAPII) and processed through subsequent steps to yield a mature mRNA that is exported from the nucleus to the cytoplasm and used by the translational machinery. The pre-mRNA undergoes several processing steps, including capping, splicing, and cleavage/polyadenylation, which appear to be precisely coordinated with nascent transcript formation (41,44,49). Of these RNA processing mechanisms, alternative splicing occurs as a widespread means to achieve proteomic diversity. Results of deep sequencing-based expression analyses estimate that more than 90% of multiexon human genes undergo alternative splicing (50, 66). The misregulation of alternative splicing underlies multiple diseases, including neurological disorders and cancer (5,19,32,67).Although transcription and alternative splicing can occur independently, both processes are physically and functionally interconnected (44, 49), and this coupling and coordination may be important for the regulation of gene expression. To date, two models have been proposed to explain the link between transcription and splicing. In the recruitment model, the unique carboxylterminal domain (CTD) of RNAPII functions as a "landing pad" for factors involved in pre-mRNA splicing in a manner that is dependent on the phosphorylation of RNAPII and the resulting functional state of the transcriptional complex (4,7,28,38,40,42,43,71). In the kinetic model, an alternative but not exclusive model, the transcript elongation...

Alternative splicing often produces effectors with opposite functions in apoptosis. Splicing decisions must therefore be tightly connected to stresses, stimuli, and pathways that control cell survival and cell growth. We have shown previously that PKC signaling prevents the production of proapoptotic Bcl-x S to favor the accumulation of the larger antiapoptotic Bcl-x L splice variant in 293 cells. Here we show that the genotoxic stress induced by oxaliplatin elicits an ATM-, CHK2-, and p53-dependent splicing switch that favors the production of the proapoptotic Bcl-x S variant. This DNA damage-induced splicing shift requires the activity of protein-tyrosine phosphatases. Interestingly, the ATM/CHK2/p53/tyrosine phosphatases pathway activated by oxaliplatin regulates Bcl-x splicing through the same regulatory sequence element (SB1) that receives signals from the PKC pathway. Convergence of the PKC and DNA damage signaling routes may control the abundance of a key splicing repressor because SB1-mediated repression is lost when protein synthesis is impaired but is rescued by blocking proteasome-mediated protein degradation. The SB1 splicing regulatory module therefore receives antagonistic signals from the PKC and the p53-dependent DNA damage response pathways to control the balance of pro-and antiapoptotic Bcl-x splice variants.

Le séquençage du génome humain a révélé que le fonctionnement biologique de l'organisme le plus complexe de la planète ne reposait que sur 25 000 gènes seulement, un nombre légèrement plus élevé que celui exprimé chez le ver Caenorhabditis elegans (environ 20 000) et considé-rablement moins grand que celui du riz (environ 35 000). Comme le nombre de gènes n'est de toute évidence pas un bon indicateur de la complexité biologique, d'autres perspectives moléculaires doivent être envisagées. L'utilisation de combinaisons précises de gènes associée à la régulation temporelle de leur expression pourrait justifier cette complexité qui caractérise les organismes supé-rieurs. De plus, un mécanisme biologique très important nommé épissage alternatif conduirait à un niveau supé-rieur de diversité et de fonctionnalité des gènes. La très grande majorité des gènes humains est constituée d'exons interrompus par des introns. Pour chacun des gènes transcrits en ARN pré-messager (pré-ARNm), l'épissage des introns permet de produire un ARN messager (ARNm) constitué d'exons dès lors positionnés de façon consécutive. L'épissage alternatif est le processus par lequel certains exons, certains introns ou des portions de ceux-ci, sont alternativement gardés ou enlevés (Figure 1). Comme plus de 70 % des gènes humains sont épissés de façon alternative [1] et que le nombre d'isoformes produits à partir d'un seul gène peut être important [2], ce processus permet à un organisme de produire un protéome beaucoup plus complexe que celui émanant d'un réservoir limité de gènes toujours épissés de façon uniforme. Bien que les cellules puissent produire une variété importante d'ARNm, des dérèglements dans les niveaux relatifs d'isoformes peuvent causer certaines maladies graves. Dans certaines situations, des mutations ciblant directement des séquences régulatrices peuvent modifier la sélection des sites d'épissage alternatif. Par exemple, des maladies telles que la déficience familiale isolée en hormone de croissance type II (IGHD II), la démence fronto-temporale avec parkinsonisme liée au chromosome 17 (FTDP-17) et la mucoviscidose sont causées par ce type d'altération [3]. De plus, un changement dans les niveaux d'expression de certains facteurs d'épissage alternatif peut influencer l'apparition de certains cancers [4,5].