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Mechanisms of Spliceosome Assembly and Splice Site Selection

剪接体组装和剪接位点选择的机制

基本信息

批准号：
8325655
负责人：
Aaron Andrew Hoskins
金额：
$ 24.83万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8325655
关键词：
3&apos Splice Site 5&apos Splice Site ATP Hydrolysis ATP phosphohydrolase Achievement Address Affect Affinity Alternative Splicing Award Base Pairing Binding Binding Proteins Biochemical Biological Assay Biological Models Biology Boxing Cell Extracts Cells Chemicals Chemistry Collaborations Complex Coupling Custom Cystic Fibrosis Disease Dissection Dissociation Distal Ensure Enzymes Event Exons Fluorescence Funding Gene Expression Regulation Genetic Goals Growth Health Hela Cells Human Human Biology Individual Instruction Introns Kinetics Laboratories Length Ligation Location Malignant Neoplasms Measurement Medicine Messenger RNA Methodology Methods Molecular Conformation Mutagenesis Mutation Nuclear Extract Organism Pathway interactions Phase Plasmids Play Positioning Attribute Probability Process Production Protein Binding Protein Conformation Protein Isoforms Proteins Publications RNA RNA Cap-Binding Proteins RNA Caps RNA Processing RNA Splicing Reaction Reading Frames Recruitment Activity Regulation Reporting Research Research Personnel Resistance Retinitis Pigmentosa Role Saccharomyces cerevisiae Scheme Science Signal Transduction Site Small Nuclear RNA Small Nuclear Ribonucleoproteins Spectrum Analysis Spliceosome Assembly Pathway Spliceosomes Staging Structure Surface System Thermodynamics Transcript U1 Small Nuclear Ribonucleoprotein U1 small nuclear RNA U2 Small Nuclear Ribonucleoprotein U2 small nuclear RNA Work Yeast Model System Yeasts base college fluorescence microscope fluorophore genetic regulatory protein human disease improved insight interest mRNA Precursor mutant prevent programs protein function protein protein interaction research study single molecule single-molecule FRET stem success tool

项目摘要

Pre-mRNA splicing is an essential step in eukaryotic gene regulation and a central process for encoding genetic complexity in higher organisms. Splicing is carried out by a MegaDalton complex of RNA and proteins called the spliceosome. Critical to the splicing process is the correct choice of the splice sites (locations of chemistry) in the pre-mRNA in order to preserve the reading frame of the transcript and produce the proper mRNA isoform by alternative splicing. The focus of this ROO application is to use single molecule fluorescence methods to elucidate the mechanisms of 5' splice site and branchsite recognition during spliceosome assembly in yeast. These mechanisms will serve as a paradigm for understanding splice site selection and alternative splicing in humans and human disease. Single molecule fluorescence methods developed during the K99 phase (see Hoskins et al., Science, 2011) allow complex reaction schemes to be dissected by following splicing pathways on individual pre-mRNAs from start to finish. These methods can be directly applied to analysis of splice site selection during the ROO phase. The 5' splice site is initially recognized by the spliceosomal U1 snRNP. The U1 snRNP engages in a number of RNA:RNA, RNA:protein, and protein:protein interactions with the pre-mRNA that all collaborate to confer affinity and fidelity. Using single molecule fluorescence, the various contributions these interactions make to the stability of the U1/5' splice site interaction will be quantified (Specific Aim 1). Auxiliary proteins often contribute to promote spliceosome assembly (e.g. splicing regulatory proteins in humans). Yeast also contain factors that can promote spliceosome assembly, and the mechanisms by which cap binding proteins promote splicing of meiotically regulated pre-mRNAs will be elucidated with single molecule methods (Specific Aim 2). Finally, correct choice ofthe branchsite by the U2 snfRNP requires ATP hydrolysis by the DEAD-box ATPase, Prp5. Single molecufe-^methods will be used to elucidate Prp5/U2/pre-mRNA interactions that promote branchsite fidelity by kinetic proofreading (Specific Aim 3).

前mrna剪接是真核生物基因调控的重要步骤，也是基因编码的核心过程