RNA. 2014 Jan;20(1):16-23. doi: 10.1261/rna.041806.113. Epub 2013 Nov 15.
The spliceosome catalyzes precursor-mRNA splicing in all eukaryotes. It consists of over 100 proteins and five small nuclear RNAs (snRNAs), including U2 and U6 snRNAs, which are essential for catalysis. Human and yeast snRNAs share structural similarities despite the fact that human snRNAs contain numerous post-transcriptional modifications. Although functions for these modifications have been proposed, their exact roles are still not well understood. To help elucidate these roles in pre-mRNA splicing, we have used single-molecule fluorescence to characterize the effect of several post-transcriptional modifications in U2 snRNA on the conformation and dynamics of the U2-U6 complex in vitro. Consistent with yeast, the human U2-U6 complex reveals the presence of a magnesium-dependent dynamic equilibrium among three conformations. Interestingly, our data show that modifications in human U2 stem I modulate the dynamic equilibrium of the U2-U6 complex by stabilizing the four-helix structure. However, the small magnitude of this effect suggests that post-transcriptional modifications in human snRNAs may have a primary role in the mediation of specific RNA-protein interactions in vivo.
剪接体在所有真核生物中催化前体 mRNA 的剪接。它由超过 100 种蛋白质和五种小核 RNA（snRNA）组成，包括 U2 和 U6 snRNA，它们对催化至关重要。尽管人类 snRNA 含有许多转录后修饰，但人类和酵母 snRNA 具有结构相似性。尽管已经提出了这些修饰的功能，但它们的确切作用仍未得到很好的理解。为了帮助阐明这些修饰在 pre-mRNA 剪接中的作用，我们使用单分子荧光技术来表征 U2 snRNA 中的几种转录后修饰对体外 U2-U6 复合物构象和动力学的影响。与酵母一致，人类 U2-U6 复合物显示出存在三种构象之间的镁依赖性动态平衡。有趣的是，我们的数据表明，人类 U2 茎 I 中的修饰通过稳定四螺旋结构来调节 U2-U6 复合物的动态平衡。然而，这种效应的幅度较小表明，人类 snRNA 中的转录后修饰可能主要在体内介导特定的 RNA-蛋白质相互作用。
