权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Administrative Supplement: Mechanisms of Spliceosome Assembly and Regulation

行政补充：剪接体组装与调控机制

基本信息

批准号：
10378361
负责人：
Aaron Andrew Hoskins
金额：
$ 16.03万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
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/10378361
关键词：
3&apos Splice Site 5&apos Splice Site ATP phosphohydrolase Active Sites Address Administrative Supplement Alternative Splicing Biochemical Chemicals Complex Cryoelectron Microscopy Disease Eukaryotic Cell Exons Fluorescence Microscopy Gene Expression Genetic Genetic Diseases Goals Human Human Genetics Introns Knowledge Label Laboratory Research Lead 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 experimental study genetic information genetic regulatory protein inhibitor/antagonist insight interdisciplinary approach mRNA Precursor preservation single molecule small molecule inhibitor

项目摘要

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?

项目总结/文摘