Antagonistic SR proteins regulate alternative splicing of tumor-related Rac1b downstream of the PI3-kinase and Wnt pathways
Antagonistic SR proteins regulate alternative splicing of tumor-related Rac1b downstream of the PI3-kinase and Wnt pathways
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DOI：
10.1093/hmg/ddp317
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发表时间：
2009-10-01
影响因子：
3.5
通讯作者：
Jordan, Peter
中科院分区：
文献类型：
--
作者：
Goncalves, Vania;Matos, Paulo;Jordan, Peter
小的GTCRac 1调节信号通路，控制肌动蛋白依赖的细胞运动以及基因转录。选择性剪接变异体Rac 1b在结直肠肿瘤亚组中过表达，并需要维持肿瘤细胞的活力。因此，了解Rac 1b通过选择性剪接过表达的分子机制具有治疗意义。在这里，我们描述了ASF/SF 2和SRp 20是两个拮抗性剪接因子调节Rac 1b在结直肠肿瘤细胞中的表达。使用Rac 1小基因，我们发现SRp 20增加了HT 29结直肠细胞中选择性外显子3b的跳跃，而ASF/SF 2增加了其包含。这些剪接因子的内源性表达的特定的小干扰RNA的耗竭证实，ASF/SF 2作为内源性Rac 1b剪接的增强子，而SRp 20作为沉默子。外显子3b中的点突变定义了两个相邻的调控区域，这两个区域是外显子3b跳跃或包含所需的，它们在体外分别被SRp 20和ASF/SF 2识别。这两种剪接因子都被发现受上游信号通路的调节：抑制磷脂酰肌醇3-激酶通路增加ASF/SF 2的蛋白水平并促进Rac 1b，而激活β-连环蛋白/TCF 4增加SRp 20的表达并抑制Rac 1b。总之，这些数据表明，信号通路协同作用，以靶向独立的剪接因子，并实现正确的组合代码，以调节小的GTTR Rac 1的选择性剪接。
The small GTPase Rac1 regulates signaling pathways controlling actin-dependent cell motility as well as gene transcription. An alternative splicing variant Rac1b is overexpressed in a subset of colorectal tumors and is required to sustain tumor cell viability. Thus, it is of therapeutic interest to understand the molecular mechanism behind the overexpression of Rac1b through alternative splicing. Here we describe that ASF/SF2 and SRp20 are two antagonistic splicing factors regulating Rac1b expression in colorectal tumor cells. Using an Rac1 minigene, we identified that SRp20 increased skipping of alternative exon 3b in HT29 colorectal cells, whereas ASF/SF2 increased its inclusion. The depletion of the endogenous expression of these splicing factors by specific small interfering RNA confirmed that ASF/SF2 acts as an enhancer of endogenous Rac1b splicing, whereas SRp20 acts as a silencer. Point mutations in exon 3b defined two adjacent regulatory regions required for skipping or inclusion of exon 3b, which are recognized in vitro by SRp20 and ASF/SF2, respectively. Both splicing factors were found to be regulated by upstream signaling pathways: the inhibition of the phosphatidylinositol 3-kinase pathway increased protein levels of ASF/SF2 and promoted Rac1b, whereas activation of beta-catenin/TCF4 increased expression of SRp20 and inhibited that of Rac1b. Together, these data reveal that signaling pathways act in concert to target independent splicing factors and achieve the correct combinatorial code to regulate alternative splicing of the small GTPase Rac1.