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: 9177613
• 负责人: JONATHAN P STALEY
• 金额: $ 34.9万
• 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 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177613
• 关键词: 3' Splice Site; ATP phosphohydrolase; Accounting; Alternative Splicing; Biochemical; Biological Assay; Biology; Blindness; Boxing; Bypass; Catalysis; Cells; Chemicals; Chemistry; Coupling; Data; Diffusion; Disease; Docking; Etiology; Gene Expression; Genes; Genetic Transcription; Genomic approach; Goals; 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; Staging; Testing; Time; Translations; U2 small nuclear RNA; Work; base; genetic approach; genome-wide; grasp; in vivo; insight; laser tweezer; mRNA Precursor; mathematical methods; mathematical model; novel; prevent; sensor; single molecule; 3' Splice Site; ATP phosphohydrolase; Accounting; Alternative Splicing; Biochemical; Biological Assay; Biology; Blindness; Boxing; Bypass; Catalysis; Cells; Chemicals; Chemistry; Coupling; Data; Diffusion; Disease; Docking; Etiology; Gene Expression; Genes; Genetic Transcription; Genomic approach; Goals; 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; Staging; Testing; Time; Translations; U2 small nuclear RNA; Work; base; genetic approach; genome-wide; grasp; in vivo; insight; laser tweezer; mRNA Precursor; mathematical methods; mathematical model; novel; 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

我们的长期目标是定义DEXD/H框ATPases在MRNA剪接中的作用，这是该的重要步骤 真核基因的表达，该基因提供了一种多功能机制，用于调节基因表达 剪接网站选择。剪接需要八个保守的DEXD/H盒ATPase。虽然认为这样的ATPases 要在几乎所有依赖RNA的过程中重塑RNA，我们对 这类分子运动蛋白很差。深入了解DEXD/H盒的作用 ATPases在剪接中，我们努力定义其RNA靶标，其重新排列RNA的机制， 以及它们在剪接站点特异性中的作用。虽然在 转录和翻译，在预先mRNA的阶段，使我们对忠诚机制的理解 仍然是基本的。虽然大多数剪接体DEXD/H BOX ATPases都涉及忠诚度，但 我们对这些ATPase如何建立剪接站点特异性的基本了解。专业 障碍是缺乏DEXD/H盒子依赖拒绝机制的测定方法。我们现在有 在剪接的催化阶段成功建立了拒绝测定法，将我们的实验室定义以定义 剪接保真度的基本基础。此外，我们最近发现这些DEXD/H盒ATPases 不仅拒绝次优的剪接站点，而且还可以启用替代方案，最佳位点，从而将这些因素定义为 在剪接催化的非常规阶段起作用的替代剪接因子。这些发现也有 对剪接体搜索3'剪接位点的基本机制产生了深入的了解。最后， 尽管在RNA依赖性过程中对DEXD/H盒ATPases的机械理解是有限的，但这些 人们通常认为蛋白质通过直接破坏RNA-RNA或RNA-蛋白质相互作用而起作用。 我们最近发现了这些ATPases间接作用的替代作用方式的证据 通过拉动一条RNA以破坏距离的相互作用。重要的是，这种新的行动方式将 允许DEXD/H BOX ATPase破坏否则将无法直接放松的双工 机制。为了在剪接的催化阶段定义DEXD/H Box ATPases的作用和机制，我们 建议完成以下三个目标，结合单分子生化， 发芽酵母中的数学，遗传和基因组方法。首先，我们旨在确定机制 并在剪接第一步进行校对的结果。其次，我们旨在定义 剪接第二步的剪接站点选择。第三，我们旨在确定DEXD/H的机制 Box ATPases在催化阶段的功能。这项工作有望改变我们对 DEXD/H BOX ATPASES中的剪接及其他。 1