Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8.
Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8.
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DOI：
10.1016/j.bbagrm.2013.11.006
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发表时间：
2014-01
期刊：
影响因子：
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通讯作者：
Shen H
中科院分区：
文献类型：
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作者：
Jang HN;Lee M;Loh TJ;Choi SW;Oh HK;Moon H;Cho S;Hong SE;Kim DH;Sheng Z;Green MR;Park D;Zheng X;Shen H
选择性剪接通过从一个基因中产生不同的蛋白质在基因表达中起着重要的作用。Caspase-2 pre-mRNA通过外显子9包合或跳脱产生抗凋亡Casp-2S和促凋亡Casp-2L蛋白。然而，外显子9剪接的分子机制尚不清楚。在这里，我们发现用siRNA敲低SRp20诱导内源性外显子9包涵显著增加。此外，SRp20的过表达促进了外显子9的跳变。因此我们得出SRp20促进外显子9跳变的结论。为了了解SRp20在caspase-2 premRNA上的功能靶点，我们对先前报道预测的SRp20潜在结合位点进行了替换和删除突变。我们证明SRp20在8外显子上的潜在结合位点的替换突变严重破坏了SRp20对9外显子跳变的影响。此外，通过RNA下拉和免疫印迹分析，我们发现SRp20与外显子8上潜在的SRp20结合RNA序列相互作用，但不与突变RNA序列相互作用。此外，我们发现从外显子8的5 ‘端删除26 nt RNA，从外显子10的3 ’端删除33 nt RNA，从内含子9删除2225 nt RNA，都不会影响SRp20在外显子9剪接上的功能。因此，我们得出结论，SRp20通过与外显子8相互作用促进caspase-2前mrna的外显子9跳跃。我们的研究结果揭示了一种新的Caspase-2 pre-mRNA剪接机制。
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 proapoptotic 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 SRp20 with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRp20 promoted exon 9 skipping. Thus we conclude that SRp20 promotes exon 9 skipping. In order to understand the functional target of SRp20 on caspase-2 premRNA, we performed substitution and deletion mutagenesis on the potential SRp20 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRp20 binding site on exon 8 severely disrupted the effects of SRp20 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRp20 interacts with the potential SRp20 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26 nt RNA from 5’ end of exon 8, a 33 nt RNA from 3’ end of exon 10 and a 2225 nt RNA from intron 9 did not compromise the function of SRp20 on exon 9 splicing. Therefore we conclude that SRp20 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.