Mechanisms of 3' Splice Site Selection in S. cerevisiae

酿酒酵母 3 剪接位点选择机制

• 批准号: 6620793
• 负责人: David Scott McPheeters
• 金额: $ 22.49万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620793
• 关键词: RNA binding protein; RNA splicing; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; active sites; adenosine triphosphate; affinity chromatography; binding sites; dinucleotide; enzyme mechanism; helicase; hydrolysis; immunoprecipitation; intermolecular interaction; introns; messenger RNA; phosphorus; point mutation; precursor mRNA; radionuclides; small nuclear RNA; small nuclear ribonucleoproteins; spliceosomes; yeasts

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.

从核前mRNAs中去除内含子需要组装一个被称为剪接体的大分子机器。据估计，在人类中，剪接体必须能够识别和精确切除多达106个不同长度和序列的不同内含子。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多个蛋白质组装到一个前-mRNA上，来精确识别内含子边界及其剪接。在许多组装步骤中，RNA螺旋在需要被称为ATP依赖的RNA解旋酶的酶的反应中形成和/或断裂。有趣的是，酵母中的遗传分析表明，RNA解旋酶的ATP水解酶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，从而确保内含子分支位点选择的保真度。最近的结果表明，Prp16对ATP的水解可能导致3‘剪接位点的结合位点的创建。拟议研究的第一个主要目标是确定Prp16p在剪接体中的RNA解旋酶活性的RNA底物，并表征由于Prp16p对ATP的水解而发生的RNA/RNA和RNA/蛋白质相互作用的重排。还将确定可能用于将Prp16p的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。其次，我们将确定Prp16p对ATP的水解是否会导致U2/U6螺旋I的相互作用发生变化，U2/U6螺旋I是第二步中具有关键功能的螺旋。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直接与Prp16p对ATP的水解有关。对RNA解旋酶家族中特定成员在剪接中的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代谢的各个方面都发挥着重要作用，这些研究的结果将与它们在其他生物环境中的功能研究相关。