Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8
Program in Gene Function and Expression; Program in Molecular Medicine
Alternative Splicing; Apoptosis; Binding Sites; Caspase 2; Exons; Gene Knockdown Techniques; HEK293 Cells; Humans; Mutagenesis; RNA Precursors; RNA-Binding Proteins; Sequence Deletion
Biochemistry | Genetics and Genomics | Molecular Biology
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.
Anti-apoptotic, Apoptotic, Caspase-2, Exon 9, Pre-mRNA splicing, SRSF3
DOI of Published Version
Jang HN, Lee M, Loh TJ, Choi SW, Oh HK, Moon H, Cho S, Hong SE, Kim do H, Sheng Z, Green MR, Park D, Zheng X, Shen H. Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8. Biochim Biophys Acta. 2014 Jan;1839(1):25-32. doi: 10.1016/j.bbagrm.2013.11.006. Link to article on publisher's site
Biochimica et biophysica acta
Jang, Ha Na; Lee, Minho; Loh, Tiing Jen; Choi, Seung-Woo; Oh, Hyun Kyung; Moon, Heegyum; Cho, Sunghee; Hong, Seong-Eui; Kim, Do Han; Sheng, Zhi; Green, Michael R.; Park, Daeho; Zheng, Xuexiu; and Shen, Haihong, "Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8" (2014). Program in Gene Function and Expression Publications and Presentations. 242.