PKC zeta controls DNA topoisomerase-dependent human caspase-2 pre-mRNA splicing.

Caspase-2 exists as two main isoforms: the caspase-2L long isoform, which is pro-apoptotic, and the caspase-2S short isoform, which may be anti-apoptotic. Topoisomerase inhibitors drive inclusion of exon 9, specific for Casp-2S mRNA, and lower Casp-2L [corrected] mRNA and protein. With cell lines engineered to express various PKC isoforms, we demonstrate that PKC zeta, but not PKCalpha, positively regulates Casp-2S mRNA assembly triggered by topoisomerase inhibitors. In addition, exon 9 inclusion is lowered in mitosis but increased in the G1/S phase. Hence, the control of caspase-2 exon 9 inclusion by topoisomerase inhibitors depends on phosphorylation and/or dephosphorylation events, and on the cell cycle phase.

Topoisomerase I and II inhibitors control caspase-2 pre-messenger RNA splicing in human cells.

We have recently shown that the topoisomerase II inhibitor, etoposide (VP16), could trigger caspase-2 pre-mRNA splicing in human leukemic cell lines. This leads to increased inclusion of exon 9, which is specifically inserted into the short caspase-2S isoform mRNA and absent from the long caspase-2L isoform mRNA. One of the consequences of this alternative splicing is a decrease in the total amount of the mature form of caspase-2L mRNA and protein. In this study, we analyzed the effects of several representative molecules of various classes of cytotoxic agents on caspase-2 pre-mRNA splicing in both U937 leukemic cells and in HeLa cervix carcinoma cells. Very strikingly, both topoisomerase I (camptothecin and homocamptothecin derivatives) and II (VP16, amsacrine, doxorubicin, mitoxantrone) inhibitors induced exon 9 inclusion. DNA intercalating glycosyl indolocarbazole derivatives as well as DNA alkylating agents, such as cisplatin and melphalan, antimetabolites like 5-fluorouracil, and mitotic spindle poisons like vinblastine had no effect. Therefore, both classes of DNA topoisomerases can control pre-mRNA splicing of the caspase-2 transcript. In addition, the splicing reaction brought about by camptothecin was hampered in human CEM/C2 and in murine P388-45R leukemic deficient in topoisomerase I activity. Conversely, VP16 did not trigger caspase-2 alternative splicing in human HL60/MX2 leukemic cells harboring a mutant topoisomerase II. Minigene transfection analysis revealed that topoisomerase inhibitors did not change the splicing profile when cis-acting elements in intron-9, reported to control exon 9 inclusion independently of drug treatment, were removed. Rather, our experiments suggest that exon 9 inclusion induced by topoisomerase inhibitors reflects the activity exerted by topoisomerase I or II on proteins that control splicing reactions, or their direct involvement in pre-mRNA splicing.

The human caspase-2 gene: alternative promoters, pre-mRNA splicing and AUG usage direct isoform-specific expression.

Caspases have been shown to play important roles in apoptotic cell death, cytokine maturation and cell differentiation. However, the transcriptional regulation of the corresponding CASP genes remains poorly known. We describe a 5.1 kb fragment located upstream of the first translated exon in the human CASP-2 gene, which is known to encode caspase-2L and -2S protein isoforms. Transient transfection experiments, together with transcription start site mapping and transcript analysis, demonstrate that each caspase mRNA is initiated from separate promoter regions, and produced from alternative splicing events in these regions. The CASP-2L promoter is much stronger than the CASP-2S promoter, in good agreement with the respective transcript levels of the two caspases. In addition, several in-frame translational start sites can be identified for each isoform, one of which is common to both, present in the second common exon, and used efficiently. Surprisingly, the short isoform may also be initiated at a downstream AUG codon within the same exon. Thus, promoter strength, alternative transcriptional initiation and 5'-splicing events regulate the expression of the main caspase-2 isoforms that may be translated from alternative translation initiation codons.