Mechanisms for Rearranging RNA during Pre-mRNA Splicing - Renewal 01

Pre-mRNA 剪接过程中 RNA 重排机制 - 更新 01

• 批准号: 9752990
• 负责人: JONATHAN P STALEY
• 金额: $ 35.48万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752990
• 关键词: 3' Splice Site; ATP phosphohydrolase; Alternative Splicing; Biochemical; Biological Assay; Biology; Blindness; Bypass; Catalysis; Cells; Chemicals; Chemistry; Coupling; Data; Diffusion; Disease; Docking; Etiology; Gene Expression; Genes; Genetic Transcription; Genomic approach; Goals; Interruption; Kinetics; Malignant Neoplasms; Modeling; Molecular; Molecular Motors; Mutation; Neurodegenerative Disorders; Positioning Attribute; Process; Proteins; RNA; RNA Binding; RNA Splicing; RNA-Protein Interaction; Reaction; Regulation; Role; Saccharomycetales; Scanning; Secure; Site; Specificity; Spliceosomes; Testing; Time; Translations; U2 small nuclear RNA; Work; genetic approach; genome-wide; grasp; in vivo; insight; laser tweezer; mRNA Precursor; mathematical methods; mathematical model; novel; nucleoside triphosphatase; prevent; sensor; single molecule

Our long-term goal is to define the role for DExD/H box ATPases in pre-mRNA splicing, an essential step in the expression of eukaryotic genes that provides a versatile mechanism for regulating gene expression through splice site choice. Splicing requires eight, conserved DExD/H box ATPases. While such ATPases are thought to remodel RNA in nearly all RNA-dependent process, our understanding of the function and mechanism of this class of molecular motor proteins is poor. Toward a deeper understanding of the role of DExD/H box ATPases in splicing, we have endeavored to define their RNA targets, their mechanisms for rearranging RNA, and their roles in splice site specificity. While fidelity mechanisms have been well-defined at the stages of transcription and translation, at the stage of pre-mRNA splicing our understanding of fidelity mechanisms remains rudimentary. While the majority of spliceosomal DExD/H box ATPases have been implicated in fidelity, we lack a fundamental understanding of how splice site specificity is established by these ATPases. A major roadblock has been the lack of assays for DExD/H box-dependent rejection mechanisms. We have now successfully established assays for rejection at the catalytic stage of splicing, positioning our lab to define the fundamental basis for splicing fidelity. Further, we have recently discovered that these DExD/H box ATPases not only reject suboptimal splice sites but also enable alternative, optimal sites, thus defining these factors as alternative splicing factors that act at the unconventional stage of splicing catalysis. These findings have also yielded insight into the fundamental mechanism by which the spliceosome searches for a 3' splice site. Lastly, while a mechanistic understanding of DExD/H box ATPases in RNA-dependent processes is limited, these proteins have been commonly thought to act by destabilizing RNA-RNA or RNA-protein interactions directly. We have recently discovered evidence for an alternative mode of action in which these ATPases act indirectly by pulling on a strand of RNA to disrupt interactions at a distance. Importantly, this new mode of action would permit a DExD/H box ATPase to disrupt a duplex that would otherwise be inaccessible for direct unwinding mechanisms. To define the role and mechanism of DExD/H box ATPases at the catalytic stage of splicing, we propose to accomplish the following three aims, using a combination of single molecule, biochemical, mathematical, genetic, and genomic approaches in budding yeast. First, we aim to determine the mechanism and consequence of proofreading at the first step of splicing. Second, we aim to define the mechanism of splice site choice at the second step of splicing. Third, we aim to determine the mechanism by which DExD/H box ATPases function at the catalytic stage. This work promises to transform our understanding of the roles of DExD/H box ATPases in splicing and beyond. 1

我们的长期目标是确定 DExD/H 盒 ATP 酶在前 mRNA 剪接中的作用，这是 真核基因的表达，提供了通过以下方式调节基因表达的通用机制 剪接位点的选择。剪接需要八个保守的 DExD/H 盒 ATP 酶。虽然此类 ATP 酶被认为 为了在几乎所有依赖RNA的过程中重塑RNA，我们对RNA的功能和机制的理解 这类分子运动蛋白很差。更深入地了解 DExD/H 盒的作用 剪接中的 ATP 酶，我们努力定义它们的 RNA 靶标、它们重排 RNA 的机制， 以及它们在剪接位点特异性中的作用。虽然保真度机制在以下阶段已经得到明确定义： 转录和翻译，在前 mRNA 剪接阶段我们对保真机制的理解 仍然处于初级阶段。虽然大多数剪接体 DExD/H 盒 ATP 酶与保真度有关， 我们对这些 ATP 酶如何建立剪接位点特异性缺乏基本的了解。一个专业 障碍是缺乏对 DExD/H 盒依赖性排斥机制的分析。我们现在有 成功建立了剪接催化阶段的排斥分析，使我们的实验室能够定义 拼接保真度的基本基础。此外，我们最近发现这些 DExD/H 盒 ATP 酶 不仅拒绝次优剪接位点，而且还启用替代的最佳剪接位点，因此将这些因素定义为 在剪接催化的非常规阶段起作用的选择性剪接因子。这些发现也 深入了解剪接体搜索 3' 剪接位点的基本机制。最后， 虽然对 RNA 依赖性过程中 DExD/H 盒 ATP 酶的机制了解有限，但这些 人们普遍认为蛋白质通过直接破坏 RNA-RNA 或 RNA-蛋白质相互作用的稳定性来发挥作用。 我们最近发现了这些 ATP 酶间接作用的替代作用模式的证据 通过拉动RNA链来破坏远距离的相互作用。重要的是，这种新的行动模式将 允许 DExD/H 盒 ATP 酶破坏双链体，否则将无法直接解旋 机制。为了明确 DExD/H 盒 ATP 酶在剪接催化阶段的作用和机制，我们 建议结合使用单分子、生化、 芽殖酵母的数学、遗传学和基因组方法。首先，我们的目标是确定机制 以及拼接第一步校对的结果。其次，我们的目标是明确机制 剪接第二步的剪接位点选择。第三，我们的目标是确定 DExD/H 的机制 盒式ATP酶在催化阶段发挥作用。这项工作有望改变我们对角色的理解 DExD/H 盒 ATP 酶在剪接及其他方面的作用。 1