Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK.
Nucleic Acids Res. 2012 Aug;40(14):6850-62. doi: 10.1093/nar/gks322. Epub 2012 Apr 13.
Spliceosomes are assembled in stages. The first stage forms complex E, which is characterized by the presence of U1 snRNPs base-paired to the 5' splice site, components recognizing the 3' splice site and proteins thought to connect them. The splice sites are held in close proximity and the pre-mRNA is committed to splicing. Despite this, the sites for splicing appear not to be fixed until the next complex (A) forms. We have investigated the reasons why 5' splice sites are not fixed in complex E, using single molecule methods to determine the stoichiometry of U1 snRNPs bound to pre-mRNA with one or two strong 5' splice sites. In complex E most transcripts with two alternative 5' splice sites were bound by two U1 snRNPs. However, the surplus U1 snRNPs were displaced during complex A formation in an ATP-dependent process requiring an intact 3' splice site. This process leaves only one U1 snRNP per complex A, regardless of the number of potential sites. We propose a mechanism for selection of the 5' splice site. Our results show that constitutive splicing components need not be present in a fixed stoichiometry in a splicing complex.
剪接体分阶段组装。第一阶段形成复杂 E，其特征是存在与 5' 剪接位点配对的 U1 snRNP，识别 3' 剪接位点的成分以及被认为连接它们的蛋白质。剪接位点紧密接近，并且前体 mRNA 被承诺进行剪接。尽管如此，似乎直到形成下一个复合物（A），剪接位点才固定。我们使用单分子方法研究了为什么在复杂 E 中 5' 剪接位点没有固定的原因，以确定具有一个或两个强 5' 剪接位点的前体 mRNA 结合的 U1 snRNP 的化学计量。在复杂 E 中，大多数具有两个替代 5' 剪接位点的转录物与两个 U1 snRNP 结合。然而，在 ATP 依赖性过程中，在复合物 A 形成过程中，多余的 U1 snRNP 被置换，该过程需要完整的 3' 剪接位点。该过程使每个复合物 A 仅留下一个 U1 snRNP，无论潜在位点的数量如何。我们提出了一种选择 5' 剪接位点的机制。我们的结果表明，组成性剪接成分不需要在剪接复合物中以固定的化学计量存在。
