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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：
8535781
负责人：
Aaron Andrew Hoskins
金额：
$ 23.62万
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/8535781
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).

前体mRNA剪接是真核生物基因调控的重要步骤，也是真核生物基因编码的核心过程。高等生物的遗传复杂性。剪接是通过RNA的兆道尔顿复合物进行的，被称为剪接体的蛋白质。剪接过程的关键是剪接位点的正确选择（化学位置）以保留转录本的阅读框架，通过选择性剪接产生合适的mRNA同种型。这个ROO应用程序的重点是使用单个用分子荧光方法阐明5'剪接位点和分支位点的识别机制在酵母中剪接体的组装过程中。这些机制将作为一个范例，剪接位点选择和可变剪接以及人类疾病。在K99阶段开发的单分子荧光方法（参见霍斯金斯等人，科学、 2011）允许通过遵循个体上的剪接途径来剖析复杂的反应方案。从开始到结束的前mRNA。这些方法可直接应用于剪接位点选择的分析在ROO阶段。5'剪接位点最初由剪接体U1 snRNP识别。的U1 snRNP参与许多RNA：RNA、RNA：蛋白质和蛋白质：蛋白质与前mRNA的相互作用所有这些共同赋予亲和力和忠诚度。使用单分子荧光，这些相互作用对U1/5'剪接位点相互作用稳定性的贡献将被量化（具体目标1）。辅助蛋白通常有助于促进剪接体组装（例如剪接人类的调节蛋白）。酵母还含有可以促进剪接体组装的因子，并且帽结合蛋白促进减数分裂调节的前mRNA剪接的机制将被用单分子方法阐明（特定目标2）。最后，正确选择分支机构的选址， U2 snfRNP需要通过DEAD盒ATP酶Prp 5水解ATP。将使用单分子方法为了阐明Prp 5/U2/pre-mRNA相互作用，通过动力学校正（Specific 目标3）。