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?

项目概要/摘要 RNA剪接是人类基因表达的一个关键特征，也是遗传扩展的主要贡献者。 选择性剪接的信息是由称为“可变剪接”的大型动态细胞机器进行的。 剪接体由组装在核上的小核糖核蛋白 (snRNP) 组成。 前体转录物（前 mRNA）去除内含子并将外显子拼接在一起，这个过程必须发生。 正是为了保存 mRNA 中携带的遗传信息，剪接的关键是正确的。 鉴定 RNA 键裂解和形成的位点 [5' 和 3' 剪接位点 (SS) 和分支 许多不同的ATP酶有助于SS和BS识别以及携带的保真度。 最近，生化研究对剪接体进行了广泛的组成和构象重塑。 通过测定酵母和人类的数十种不同结构，剪接的方式发生了变化 尽管发生了这种结构革命，但关于剪接体的核心特征仍然有很多未知之处。 我实验室的研究目标是阐明剪接体的机制。 我们经常使用单分子进行生化深度组装和调节。 荧光显微镜对复杂且异质的反应途径进行解卷积 在最近的工作中，我们研究了5'SS和BS的识别、组装和机制。 U6 snRNP 的动力学，并开发了荧光标记、纯化和抑制 RNA 的方法 我们未来五年的愿景是融合从剪接体结构中获得的见解。 通过单分子、生化、计算和遗传实验来解决我们的突出差距 这些差距包括 RNP 折叠和组装的基本原理、 调控剪接的机制，以及缺乏特异性和有效的化学抑制剂 作为这一愿景的一部分，我们将使用多学科方法回答以下问题： 1) RNA和蛋白质如何共折叠组装U6 snRNP？ 2）剪接体在其活性位点创建过程中是如何重塑的？ 3) 调节蛋白如何促进弱 5'SS 处的剪接？ 4) 我们如何用小分子抑制剂阻断剪接过程中 ATP 酶依赖性转变？ 5）我们如何定量分析、比较和整合剪接体的冷冻电镜结构？