Regulation of alternative splicing of caspase-2 through an intracellular signaling pathway in response to pro-apoptotic stimuli

Alternative splicing is an important mechanism in the generation of functionally distinct products from the same gene. Some apoptosis-regulating genes also undergo alternative splicing, generating splice variants that antagonzie normal transcripts on apoptosis. For example, caspase-2 is alternatively spliced, leading to exon 9–lacking caspase-2L (proapoptotic) and exon 9–containing caspase-2S (antiapoptotic) transcripts. Serine-arginine splicing factor proteins (SR proteins) are highly conserved and required for constitutive and alternative messenger RNA (mRNA) splicing. Their activity is regulated by reversible phosphorylation on serine residue. During apoptosis, many functional molecules undergo posttranslational modification, including phosphorylation, dephosphorylation, and caspase cleavage. In this study, we investigated the effect of proapoptotic stimuli on alternative splicing of caspase-2 mRNA in U937 cells. U937 cells were simulated with etoposide, staurosporine, pacritaxel, or cyclohexamide. We analzyed the alternative splicing of caspase-2 mRNA using reverse transcription-polymerase chain reaction. Etoposide, staurosporine, pacritaxel, and cyclohexamide treatment promoted exon-9 inclusion, increasing the ratio of caspase-2S to caspase-2L in a time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase-1, blocked etoposide-induced alternative splicing of caspase-2 mRNA. Furthermore, pretreatment of U937 cells with fumonisin B1, an inhibitor of ceramide synthase, also blocked alternative splicing of caspase-2 mRNA. These data demonstrate that endogenous ceramide generation and subsequent phosphatase activation during apoptosis are key steps in the alternative splicing of caspase-2 mRNA and further suggest a link between the signal-transduction pathway and alternative splicing.

Abbreviations:  ASF/SF2, alternative splicing factor/splicing factor-2 , ΔΨm, loss of mitochondrial membrane potential , GAPDH, reduced glyceraldehyde-phosphate dehydrogenase , hnRNPA1, heterogeneous nuclear ribonucleoprotein A1 , JNK, c-Jun N-terminal kinase , MAPK, mitogen-activated protein kinase , RT-PCR, reverse transcription-polymerase chain reaction , SR protein, serine-arginine protein