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.

See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.

SummaryRNA binding proteins and signaling components control the production of pro-death and pro-survival splice variants of Bcl-x. DNA damage promoted by oxaliplatin increases the level of pro-apoptotic Bcl-xS in an ATM/CHK2-dependent manner, but how this shift is enforced is not known. Here, we show that in normally growing cells, when the 5′ splice site of Bcl-xS is largely repressed, SRSF10 partially relieves repression and interacts with repressor hnRNP K and stimulatory hnRNP F/H proteins. Oxaliplatin abrogates the interaction of SRSF10 with hnRNP F/H and decreases the association of SRSF10 and hnRNP K with the Bcl-x pre-mRNA. Dephosphorylation of SRSF10 is linked with these changes. A broader analysis reveals that DNA damage co-opts SRSF10 to control splicing decisions in transcripts encoding components involved in DNA repair, cell-cycle control, and apoptosis. DNA damage therefore alters the interactions between splicing regulators to elicit a splicing response that determines cell fate.

The alternative splicing of Bcl-x generates the proapoptotic Bcl-x S protein and the antiapoptotic isoform Bcl-x L . Bcl-x splicing is coupled to signal transduction, since ceramide, hormones, and growth factors alter the ratio of the Bcl-x isoforms in different cell lines. Here we report that the protein kinase C (PKC) inhibitor and apoptotic inducer staurosporine switches the production of Bcl-x towards the x S mRNA isoform in 293 cells. The increase in Bcl-x S elicited by staurosporine likely involves signaling events that affect splicing decisions, because it requires active transcription and no new protein synthesis and is independent of caspase activation. Moreover, the increase in Bcl-x S is reproduced with more specific inhibitors of PKC. Alternative splicing of the receptor tyrosine kinase gene Axl is similarly affected by staurosporine in 293 cells. In contrast to the case for 293 cells, PKC inhibitors do not influence the alternative splicing of Bcl-x and Axl in cancer cell lines, suggesting that these cells have sustained alterations that uncouple splicing decisions from PKC-dependent signaling. Using minigenes, we show that an exonic region located upstream of the Bcl-x S 5 splice site is important to mediate the staurosporine shift in Bcl-x splicing. When transplanted to other alternative splicing units, portions of this region confer splicing modulation and responsiveness to staurosporine, suggesting the existence of factors that couple splicing decisions with PKC signaling.Alternative splicing of pre-mRNAs provides a powerful mechanism to augment the protein repertoire encoded by metazoan genomes. It is estimated that 74% of all multiexonic human genes are alternatively spliced (36). Moreover, alternative splicing is becoming increasingly relevant to a variety of human diseases, including cancer (60, 85). These observations justify current efforts devoted at uncovering the basic principles of alternative splicing control. Many studies have provided valuable insights into the roles of specific elements and factors in splicing modulation (7,58,84). Proteins that bind to specific sequence elements to affect splice site selection include SR proteins, hnRNP proteins, and other related RNA binding proteins such as TIA-1, ETR-3, and Sam68 (16,29,35,54,59,76). Many of these proteins can be modified posttranslationally, and some of the modifications, like phosphorylation, can affect their localization and activity (59, 82).Although we expect that the coupling between signal transduction events and alternative splicing decisions will represent a major network of regulation, very little is known about how splicing is coordinated by a variety of effectors to attune cells to specific environmental demands and stresses. Possibly the first system describing a link between signal transduction and splicing involved the cell surface molecule CD44, in which exon v5 inclusion was stimulated following activation of the protein kinase C (PKC) or extracellular signal-regulated kinase, as part of the Ras signaling path...

The Bcl-x pre-mRNA is alternatively spliced to produce the anti-apoptotic Bcl-x L and the pro-apoptotic Bcl-x S isoforms. By performing deletion mutagenesis on a human Bcl-x minigene, we have identified a novel exonic element that controls the use of the 5 splice site of Bcl-x S . The proximal portion of this element acts as a repressor and is located downstream of an enhancer. Further mutational analysis provided a detailed topological map of the regulatory activities revealing a sharp transition between enhancer and repressor sequences. Portions of the enhancer can function when transplanted in another alternative splicing unit. Chromatography and immunoprecipitation assays indicate that the silencer element interacts with heterogeneous ribonucleoprotein particle (hnRNP) K, consistent with the presence of putative high affinity sites for this protein.Finally, down-regulation of hnRNP K by RNA interference enhanced splicing to Bcl-x S , an effect seen only when the sequences bound by hnRNP K are present. Our results therefore document a clear role for hnRNP K in preventing the production of the pro-apoptotic Bcl-x S splice isoform.Alternative splicing is a major mechanism used to augment the number of proteins encoded by the genome. It is estimated that as many as 97% of multiple exon pre-mRNAs undergo alternative splicing (1, 2). Disruption of alternative splicing by mutating important regulatory sequences or by altering the expression or activity of proteins controlling splice site selection has been linked with different diseases, including cancer (3-7). Apoptosis is an important and complex cellular program involved in development and differentiation in higher organisms (8, 9). However, its aberrant control often contributes to cancer development and the resistance of cancer cells to drug therapy (10 -13).Genes implicated in the apoptotic pathway are alternatively spliced often to produce protein isoforms with distinct or even antagonistic activities (14,15). A good example is the apoptotic regulator Bcl-x, which is alternatively spliced to produce two major isoforms, the anti-apoptotic Bcl-x L protein and the shorter pro-apoptotic Bcl-x S isoform (16). This alternative splicing decision involves a competition between two 5Ј splice sites; the use of the downstream site creates Bcl-x L , and the use of the upstream one produces Bcl-x S (Fig. 1A). Bcl-x L is always the predominant form in cancer cells, and overexpressing it can confer resistance to chemotherapeutic agents (17-22). On the other hand, overexpression of the pro-apoptotic Bcl-x S isoform enhances sensitivity to the topoisomerase inhibitor etoposide and to taxol in a breast cancer cell line, while triggering apoptosis in melanoma cell lines (23,24). Using antisense technologies to improve the production of the Bcl-x S splice variant can also induce apoptosis in cancer cells (25)(26)(27).Alternative splicing is regulated by different proteins bound to sequence elements near splice sites. A variety of mechanisms is used to achieve regulation. Some s...