Johanne Toutant

Proteins Associated with the Exon Junction Complex Also Control the Alternative Splicing of Apoptotic Regulators

ABSTRACT 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-xS 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.

Anticancer drugs affect the alternative splicing of Bcl-x and other human apoptotic genes

Inducing an apoptotic response is the goal of most current chemotherapeutic interventions against cancer. However, little is known about the effect of chemotherapeutic agents on the alternative splicing of apoptotic genes. Here, we have tested 20 of the mainstream anticancer drugs for their ability to influence the production of Bcl-x splice isoforms. We find that many drugs shift splicing toward the proapoptotic Bcl-xS splice variant in 293 cells. The drugs modulate splicing decisions most likely through signaling events because the splicing switch is not compromised by inhibiting de novo protein synthesis or the activity of caspases. Several drugs also shift Bcl-x splicing in cancer cell lines (MCF-7, HeLa, PC-3, PA-1, and SKOV-3), but the set of active drugs varies between cell lines. We also examined the effect of anticancer agents on the alternative splicing of 95 other human apoptotic genes in different cell lines. Almost every drug can alter a subset of alternative splicing events in each cell line. Although drugs of the same class often influence the alternative splicing of the same units in individual cell lines, these units differ considerably between cell lines, indicating cell line–specific differences in the pathways that control splicing. [Mol Cancer Ther 2008;7(6):1398–409]

Heterogeneous Nuclear Ribonucleoprotein K Represses the Production of Pro-apoptotic Bcl-xS Splice Isoform

The Bcl-x pre-mRNA is alternatively spliced to produce the anti-apoptotic Bcl-xL and the pro-apoptotic Bcl-xS 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-xS. 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-xS, 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-xS splice isoform.

Protein kinase C-dependent control of Bcl-x alternative splicing.

ABSTRACT The alternative splicing of Bcl-x generates the proapoptotic Bcl-xS protein and the antiapoptotic isoform Bcl-xL. 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 xS mRNA isoform in 293 cells. The increase in Bcl-xS 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-xS 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-xS 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.

Antagonistic Effects of the SRp30c Protein and Cryptic 5 ′ Splice Sites on the Alternative Splicing of the Apoptotic Regulator Bcl-x

Alternative 5 ′ splice site selection allows Bcl-x to produce two isoforms with opposite effects on apoptosis. The pro-apoptotic Bcl-xS variant is up-regulated by ceramide and down-regulated by protein kinase C through specific cis-acting exonic elements, one of which is bound by SAP155. Splicing to the Bcl-xS 5 ′ splice site is also enforced by heterogeneous nuclear ribonucleoprotein (hnRNP) F/H proteins and by Sam68 in cooperation with hnRNP A1. Here, we have characterized exon elements that influence splicing to the 5 ′ splice site of the anti-apoptotic Bcl-xL isoform. Within a 86-nucleotide region (B3) located immediately upstream of the Bcl-xL donor site we have identified two elements (ML2 and AM2) that stimulate splicing to the Bcl-xL 5 ′ splice site. SRp30c binds to these elements and can shift splicing to the 5 ′ splice site of Bcl-xL in an ML2/AM2-dependent manner in vitro and in vivo. The B3 region also contains an element that represses the use of Bcl-xL. This element is bound by U1 small nuclear ribonucleoprotein and contains two 5 ′ splice sites that can be used when the Bcl-xL 5 ′ splice site is mutated or the ML2/AM2 elements are deleted. Conversely, mutating the cryptic 5 ′ splice sites stimulates splicing to the Bcl-xL site. Thus, SRp30c stimulates splicing to the downstream 5 ′ splice site of Bcl-xL, thereby attenuating the repressive effect of upstream U1 snRNP binding sites.

The DNA Damage Response Pathway Regulates the Alternative Splicing of the Apoptotic Mediator Bcl-x

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-xS to favor the accumulation of the larger antiapoptotic Bcl-xL 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-xS 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.

RBFOX1 Cooperates with MBNL1 to Control Splicing in Muscle, Including Events Altered in Myotonic Dystrophy Type 1

With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1.

SRSF10 Connects DNA Damage to the Alternative Splicing of Transcripts Encoding Apoptosis, Cell-Cycle Control, and DNA Repair Factors.

Summary RNA 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.

TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis

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.

Modulation of the splicing regulatory function of SRSF10 by a novel compound that impairs HIV-1 replication

Abstract We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro. Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2β, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2β, concentrations greater than those needed to inhibit HIV-1 replication were required to elicit significant alterations. Thus, the ability of 1C8 to alter the SRSF10-dependent splicing of HIV-1 transcripts, with minor effects on cellular splicing, supports the view that SRSF10 may be used as a target for the development of new anti-viral agents.