Dietrich R C, Shukla G C, Fuller J D, Padgett R A
Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
RNA. 2001 Oct;7(10):1378-88.
Alternative splicing increases the coding capacity of genes through the production of multiple protein isoforms by the conditional use of splice sites and exons. Many alternative splice sites are regulated by the presence of purine-rich splicing enhancer elements (ESEs) located in the downstream exon. Although the role of ESEs in alternative splicing of the major class U2-dependent introns is well established, no alternatively spliced minor class U12-dependent introns have so far been described. Although in vitro studies have shown that ESEs can stimulate splicing of individual U12-dependent introns, there is no direct evidence that the U12-dependent splicing system can respond to ESEs in vivo. To investigate the ability of U12-dependent introns to use alternative splice sites and to respond to ESEs in an in vivo context, we have constructed two sets of artificial minigenes with alternative splicing pathways and evaluated the effects of ESEs on their alternative splicing patterns. In minigenes with alternative U12-dependent 3' splice sites, a purine-rich ESE promotes splicing to the immediately upstream 3' splice site. As a control, a mutant ESE has no stimulatory effect. In minigene constructs with two adjacent U12-dependent introns, the predominant in vivo splicing pattern results in the skipping of the internal exon. Insertion of a purine-rich ESE into the internal exon promotes the inclusion of the internal exon. These results show that U12-dependent introns can participate in alternative splicing pathways and that U12-dependent splice sites can respond to enhancer elements in vivo.
可变剪接通过有条件地使用剪接位点和外显子产生多种蛋白质异构体，从而增加基因的编码能力。许多可变剪接位点受位于下游外显子中的富含嘌呤的剪接增强子元件（ESE）的调控。尽管ESE在主要类型的U2依赖性内含子的可变剪接中的作用已得到充分证实，但迄今为止尚未描述过可变剪接的次要类型U12依赖性内含子。虽然体外研究表明ESE可以刺激单个U12依赖性内含子的剪接，但没有直接证据表明U12依赖性剪接系统在体内能够对ESE作出反应。为了研究U12依赖性内含子在体内环境中使用可变剪接位点以及对ESE作出反应的能力，我们构建了两组具有可变剪接途径的人工微型基因，并评估了ESE对其可变剪接模式的影响。在具有可变U12依赖性3'剪接位点的微型基因中，富含嘌呤的ESE促进剪接至紧邻的上游3'剪接位点。作为对照，突变的ESE没有刺激作用。在具有两个相邻U12依赖性内含子的微型基因构建体中，体内主要的剪接模式导致内部外显子的跳跃。将富含嘌呤的ESE插入内部外显子可促进内部外显子的包含。这些结果表明，U12依赖性内含子可以参与可变剪接途径，并且U12依赖性剪接位点在体内能够对增强子元件作出反应。
