SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness

Abstract

Two splice variants are derived from the caspase-9 gene, proapoptotic caspase-9a and antiapoptotic caspase9b, by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. Previous studies from our laboratory have shown that the alternative splicing of caspase-9 and the phosphorylation status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and ceramide via the action of protein phosphatase-1. In this study, a link between ceramide, SR proteins, and the alternative splicing of caspase-9 was established. The downregulation of SRp30a in A549 cells by RNA interference technology resulted in an increase in the caspase-9b splice variant, with a concomitant decrease in the caspase-9a splice variant, thereby significantly decreasing the caspase-9a/9b ratio from 1.67 6 0.11 to 0.56 6 0.08 (P , 0.005). The specific downregulation of SRp30a also inhibited the ability of exogenous ceramide treatment to induce the inclusion of the exon 3, 4, 5, and 6 cassette. Therefore, we have identified SRp30a as an RNA trans-acting factor that functions as a major regulator of caspase-9 pre-mRNA processing and is required for ceramide to mediate the alternative splicing of caspase-9.-Massiello, A., and C. E. Chalfant. SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness. J. Lipid Res. 2006. 47: 892-897. Supplementary key words RNA cis-element . RNA trans-acting factor . A549 cellsCeramide is an important regulator of various stress responses and growth mechanisms, and the formation of ceramide from the hydrolysis of sphingomyelin or from de novo pathways has been observed in response to agonists such as tumor necrosis factor-a, g-interferon, 1a,25-dihydroxyvitamin D 3 , interleukin-1, ultraviolet light, heat, chemotherapeutic agents, fatty acid synthase antigen, and nerve growth factor (1-7). Also, the addition of exogenous ceramide or the enhancement of cellular levels of ceramide induces cell differentiation, cell cycle arrest, apoptosis, or cell senescence in various cell types (8-10). The prominent role of ceramide as a regulator of cellular mechanisms necessitated the identification of target molecules. To this end, a family of ceramide-regulated enzymes has been identified, ceramide-activated protein phosphatases, which include the serine/threonine-specific protein phosphatases PP1 and PP2A (11)(12)(13)(14). With the demonstration of PP1 as a ceramide-activated protein phosphatase, potential PP1 substrates and mechanisms regulated by PP1 became candidate targets for ceramide action.SR proteins, a family of arginine/serine-rich domaincontaining proteins and specific PP1 substrates, are required for constitutive and alternative pre-mRNA processing. Endogenous ceramide was recently found to modulate the phosphorylation status of SR proteins in a PP1-dependent manner (15). Several reports have also demonstrated a role for PP1 in regulating alternative splicing, and two spliceosomal targeting subunits for PP1 ha...

The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer

“…For example, SRp30a (SRSF1) was shown to be a proto-oncogene and up-regulated in a large percentage of breast cancers (39 -41). This factor has also been shown to be hyperphosphorylated in lung cancer cells, which is linked to the alternative splicing of caspase 9 and cancer phenotypes such as anchorageindependent growth, chemotherapy resistance, and tumor formation (42,43). The expression of a number of RNA splicing factors is also linked to tumor progression in various cancers and cancer phenotypes.…”

Alternative Splicing Factor/Splicing Factor 2 Regulates the Expression of the ζ Subunit of the Human T Cell Receptor-associated CD3 Complex

“…The splicing regulatory activity of ASF/ SF2 depends on its phosphorylation status, and the sphingolipid metabolite D-erythro-C 6 -ceramide is shown to modulate the splicing activity of ASF/SF2 through a protein phosphatase 1-mediated dephosphorylation of ASF/SF2 (19). We treated T cells with increasing concentrations of ceramide and assessed its effect on CD3 UTR alternative splicing by reverse transcription (RT)-PCR.…”

E2F1 controls alternative splicing pattern of genes involved in apoptosis through upregulation of the splicing factor SC35

“…[15][16] The inclusion or exclusion of an exon cassette in caspase-9 causes the expression of two splice variants, namely the pro-apoptotic caspase-9a and antiapoptotic caspase-9b. [17][18] To assess whether E2F1 induces changes in the alternative splicing profile of these caspases pre-mRNAs, RNAs recovered from non-induced or induced H358/Tet-On/E2F1 cells were analyzed by RT-PCR using primers specific for each caspase splice variant (Figure 4a). The results showed that E2F1 increased the expression of pro-apoptotic caspases-2L, -8a and -9a mRNA levels and decreased those of anti-apoptotic caspases-8L and -9b (Figure 4b).…”