Institute of Biochemistry, Justus Liebig University of Giessen, Giessen, Germany.
EMBO J. 2011 May 18;30(10):1965-76. doi: 10.1038/emboj.2011.106. Epub 2011 Apr 5.
Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.
精确的 5'剪接位点识别对于组成型和调节性前体 mRNA 剪接都是必不可少的。U1 小核核糖核蛋白颗粒（snRNP）特异性蛋白 U1C 参与剪接体组装的这第一步，对于稳定早期剪接复合物很重要。我们使用一种胚胎致死的 U1C 突变斑马鱼 hi1371 来研究 U1C 对剪接调控的潜在全基因组作用。U1C 突变体胚胎包含总体稳定的，但 U1C 缺失的 U1 snRNPs。令人惊讶的是，突变体与野生型胚胎的全基因组 RNA-Seq 分析显示了一大组特定的靶基因，它们在缺乏 U1C 的情况下改变了它们的可变剪接模式。将 ZfU1C cRNA 注射到突变体胚胎中，并在 HeLa 细胞中进行 siRNA 介导的 U1C 敲低后的体内剪接实验，证实了 U1C 的依赖性和特异性，以及观察到的效应的功能保守性。此外，对 U1C 依赖性 5'剪接位点的序列模体分析揭示了与下游内含子 U 丰富元件的关联。总之，我们的发现为一种普遍的 snRNP 蛋白 U1C 作为可变剪接调控的介质提供了新的证据。
