Mechanisms of 3' Splice Site Selection in S. cerevisiae

从核前mrna中去除内含子需要组装一个称为剪接体的大型大分子机器。据估计，在人类中，剪接体必须能够识别并精确切除多达106个不同长度和序列的内含子。剪接体通过高度有序和动态地将5个小核核糖-核蛋白颗粒和60多种蛋白质组装到mrna前体上，精确地识别内含子边界并对其进行剪切。在许多组装步骤中，RNA螺旋形成和/或断裂的反应需要称为ATP依赖性RNA解旋酶的酶。有趣的是，酵母的遗传分析表明，RNA解旋酶水解atp可以在校对步骤中发挥作用，确保剪接位点选择的保真度。Prp16p是一种RNA解旋酶，它催化剪接体的构象变化以确保内含子分支位点选择的保真度。最近的研究结果表明，Prp16对ATP的水解可能导致3'剪接位点的结合位点的产生。本研究的第一个主要目标是确定剪接体中Prp16p解旋酶活性的RNA底物，并表征由于ATP被Prp16p水解而发生的RNA/RNA和RNA/蛋白质相互作用的重排。Prp16p将ATP水解与分支位点识别的保真度联系起来的相互作用的性质也将被确定。其次，我们将确定Prp16p水解ATP是否会导致U2/U6螺旋I的相互作用发生变化，这一螺旋在第二步中具有关键功能。第三，由于Prp16p促进的重排可能最终形成3‘剪接位点的结合位点，我们将确定涉及3’剪接位点的相互作用的变化是否与Prp16p对ATP的水解直接相关。对RNA解旋酶家族中一个特定成员在剪接中所起作用的详细描述应该有助于阐明它们发挥作用的原理。由于这些酶在细胞RNA代谢的各个方面都起着重要的作用，因此这些研究的结果将与研究它们在其他生物学背景下的功能有关。

The removal of introns from nuclear pre-mRNAs requires the assembly of a large macromolecular machine termed the spliceosome. In humans, it has been estimated that the spliceosome must be able to recognize and precisely excise as many as 106 different introns of varying lengths and sequences. The precise identification of intron boundaries and their excision by the spliceosome is accomplished through the highly ordered and dynamic assembly of five small nuclear ribonucleoprotein particles and more than sixty proteins onto a pre-mRNA. During many of the assembly steps, RNA helices are formed and/or broken in reactions requiring enzymes termed ATP- dependent RNA helicases. Intriguingly, genetic analyses in yeast have indicated that ATP-hydrolysis by RNA helicases can function in proofreading steps that insure the fidelity of splice site choice. Prp16p is an RNA helicase that catalyzes a conformational change in the spliceosome acts to insure the fidelity of intron branch site selection. Recent results have suggested that ATP hydrolysis by Prp16 may lead to the creation of a binding site for the 3' splice site. The first major goal of the proposed research is to identify the RNA substrates for the RNA helicase activity of Prp16p within the spliceosome, and to characterize the rearrangements of RNA/RNA and RNA/protein interactions that occur as a consequence of ATP hydrolysis by Prp16p. The nature of the interactions that may serve to connect ATP hydrolysis by Prp16p to the fidelity of branch site recognition will also be determined. Second, we will determine if ATP hydrolysis by Prp16p leads to changes in the interactions of U2/U6 helix I, a helix that has a critical function in the second step. Third, because the rearrangements facilitated by Prp16p may culminate in the formation of a binding site for the 3' splice site, we will determine if changes in the interactions involving the 3' splice site are directly tied to ATP hydrolysis by Prp16p. A detailed picture of the role of a specific member of the RNA helicase family in splicing should help elucidate the principles by which they function. Because these enzymes have important roles in all aspects of cellular RNA metabolism, the results of these studies will be relevant to the study of their functions in other biological contexts.