Simard M J, Chabot B
Département de Microbiologie et d'Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.
Mol Cell Biol. 2000 Oct;20(19):7353-62. doi: 10.1128/MCB.20.19.7353-7362.2000.
Alternative splicing of exon 7B in the hnRNP A1 pre-mRNA produces mRNAs encoding two proteins: hnRNP A1 and the less abundant A1B. We have reported the identification of several intron elements that contribute to exon 7B skipping. In this study, we report the activity of a novel element, conserved element 9 (CE9), located in the intron downstream of exon 7B. We show that multiple copies of CE9 inhibit exon 7B-exon 8 splicing in vitro. When CE9 is inserted between two competing 3' splice sites, a single copy of CE9 decreases splicing to the distal 3' splice site. Our in vivo results also support the conclusion that CE9 is a splicing modulator. First, inserting multiple copies of CE9 into an A1 minigene compromises the production of fully spliced products. Second, one copy of CE9 stimulates the inclusion of a short internal exon in a derivative of the human beta-globin gene. In this case, in vitro splicing assays suggest that CE9 decreases splicing of intron 1, an event that improves splicing of intron 2 and decreases skipping of the short internal exon. The ability of CE9 to act on heterologous substrates, combined with the results of a competition assay, suggest that the activity of CE9 is mediated by a trans-acting factor. Our results indicate that CE9 represses the use of the common 3' splice site in the hnRNP A1 alternative splicing unit.
hnRNP A1前体mRNA中外显子7B的可变剪接产生编码两种蛋白质的mRNA：hnRNP A1和丰度较低的A1B。我们已报道了几种有助于外显子7B跳跃的内含子元件的鉴定。在本研究中，我们报道了位于外显子7B下游内含子中的一种新型元件保守元件9（CE9）的活性。我们表明，多个CE9拷贝在体外抑制外显子7B-外显子8的剪接。当CE9插入两个相互竞争的3'剪接位点之间时，单个CE9拷贝会减少向远端3'剪接位点的剪接。我们的体内结果也支持CE9是一种剪接调节因子的结论。首先，将多个CE9拷贝插入A1微型基因会损害完全剪接产物的产生。其次，一个CE9拷贝会刺激人β-珠蛋白基因衍生物中一个短内部外显子的包含。在这种情况下，体外剪接试验表明CE9会减少内含子1的剪接，这一事件会改善内含子2的剪接并减少短内部外显子的跳跃。CE9作用于异源底物的能力，结合竞争试验的结果，表明CE9的活性是由一种反式作用因子介导的。我们的结果表明，CE9在hnRNP A1可变剪接单元中抑制常见3'剪接位点的使用。
