Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8.

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5' end of exon 8, a 33nt RNA from 3' end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.