Mechanisms of Spliceosome Assembly and Regulation

剪接体组装和调控机制

• 批准号: 10608952
• 负责人: Aaron Andrew Hoskins
• 金额: $ 37.31万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608952
• 关键词: 3' Splice Site; 5' Splice Site; ATP phosphohydrolase; Active Sites; Address; Alternative Splicing; Biochemical; Chemicals; Complex; Cryoelectron Microscopy; Disease; Eukaryotic Cell; Exons; Experimental Genetics; Fluorescence Microscopy; Gene Expression; Genetic Diseases; Goals; Human; Human Genetics; Introns; Knowledge; Label; Laboratory Research; Messenger RNA; Methods; Molecular Conformation; Nuclear RNA; Pathology; Pathway interactions; Process; Proteins; RNA; RNA Splicing; Reaction; Regulation; Role; Site; Small Nuclear Ribonucleoproteins; Spliceosome Assembly Pathway; Spliceosomes; Structure; Techniques; Transcript; U6 Small Nuclear Ribonucleoproteins; Vision; Work; Yeasts; genetic information; genetic regulatory protein; inhibitor; insight; interdisciplinary approach; mRNA Precursor; preservation; single molecule; small molecule inhibitor

PROJECT SUMMARY/ABSTRACT RNA splicing is a key feature of human gene expression and a major contributor to expansion of genetic information by alternative splicing. Splicing is carried out by a large and dynamic cellular machine called the spliceosome. Spliceosomes are composed of small nuclear ribonucleoproteins (snRNPs) that assemble on precursor transcripts (pre-mRNAs) to remove introns and splice together exons. This process must occur precisely in order to preserve the genetic information carried in the mRNA. Critical for splicing is the correct identification of the sites of RNA bond cleavage and formation [the 5' and 3' splice sites (SS) and the branch site (BS)]. A number of different ATPases contribute to the fidelity of SS and BS recognition as well as carry out extensive compositional and conformational remodeling of the spliceosome. Recently, biochemical studies of splicing have been transformed by determination of dozens of different structures of yeast and human spliceosomes by cryo-EM. Despite this structural revolution, much remains unknown about central features of the splicing reaction. The goal of my laboratory’s research is to elucidate mechanisms of spliceosome assembly and regulation in biochemical depth using a variety of techniques. We often use single molecule fluorescence microscopy to deconvolute the complex and heterogeneous reaction pathways employed by the splicing machinery. In recent work, we have studied mechanisms of 5'SS and BS recognition, assembly and dynamics of the U6 snRNP, and developed methods for fluorescently-labeling, purifying, and inhibiting RNAs and RNPs. Our vision for the next five years is to merge the insights obtained from structures of spliceosomes with single molecule, biochemical, computational, and genetic experiments to address outstanding gaps in our knowledge of splicing. These gaps include fundamental principles of RNP folding and assembly, the mechanisms of regulated splicing, and the scarcity of specific and effective chemical inhibitors of the spliceosome. As part of this vision, we will answer the following questions using multi-disciplinary approaches: 1) How do RNA and protein co-fold to assemble the U6 snRNP? 2) How is the spliceosome remodeled during creation of its active site? 3) How do regulatory proteins promote splicing at weak 5'SS? 4) How can we block ATPase-dependent transitions during splicing with small molecule inhibitors? 5) How do we quantitatively analyze, compare, and integrate cryo-EM structures of spliceosomes?

项目摘要/摘要 RNA剪接是人类基因表达的关键特征，也是遗传扩展的主要促进者 通过替代剪接信息。拼接是由一台称为The的大型动态蜂窝机进行的 剪接体。剪接体由组装在上的小核核糖核蛋白（SNRNP）组成 前体转录本（PRE-MRNA）去除内含子并剪接出外显子。这个过程必须发生 正是为了保留mRNA中携带的遗传信息。剪接至关重要的是正确的 RNA键裂解和形成的位点[5'和3'剪接位点（SS）和分支 站点（BS）]。许多不同的ATP酶有助于SS和BS识别的忠诚以及随身携带 消除剪接体的广泛组成和构象重塑。最近，生化研究 通过确定数十种酵母和人类的不同结构来改变剪接 Cryo-Em的剪接体。尽管进行了这场结构性革命，但关于中心特征的许多尚不清楚 剪接反应。我实验室研究的目的是阐明剪接体的机制 使用多种技术的生化深度组装和调节。我们经常使用单分子 荧光显微镜以否定了由该复合物和异质反应途径 剪接机械。在最近的工作中，我们研究了5'S和BS识别，组装和BS的机制 U6 SNRNP的动力学，并开发了用于荧光标记，净化和抑制RNA的方法 和RNP。我们接下来五年的愿景是合并从剪接结构中获得的见解 使用单分子，生化，计算和遗传实验，以解决我们的杰出差距 剪接知识。这些差距包括RNP折叠和组装的基本原理， 受调节剪接的机制，以及特定有效的化学抑制剂的稀缺性 剪接体。作为该愿景的一部分，我们将使用多学科方法回答以下问题： 1）RNA和蛋白质如何共同组装U6 SNRNP？ 2）在创建其活性站点期间，剪接体如何重塑？ 3）调节蛋白如何在弱5'S上促进剪接？ 4）如何使用小分子抑制剂剪接过程中ATPase依赖性过渡？ 5）我们如何定量分析，比较和整合剪接体的冷冻EM结构？