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

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

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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剪接是真核基因调节的重要步骤，也是编码的中心过程较高生物体中的遗传复杂性。剪接由RNA的Megadalton建筑群进行蛋白质称为剪接体。对剪接过程至关重要的是剪接站点的正确选择（化学位置）在前MRNA中，以保留成绩单的阅读框通过替代剪接产生适当的mRNA同工型。此Roo应用程序的重点是使用单个分子荧光方法阐明了5'剪接位点和分支识别的机制在酵母中的剪接组装过程中。这些机制将成为理解的范式人类和人类疾病中的剪接部位选择和替代剪接。在K99阶段开发的单分子荧光方法（参见Hoskins等，Science， 2011年）允许通过遵循单个的拼接途径来解剖复杂的反应方案从头到尾。这些方法可以直接应用于剪接站点选择的分析在Roo阶段。最初，5'剪接位点由剪接体U1 SNRNP识别。 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都合作赋予亲和力和忠诚。使用单分子荧光，各种这些相互作用对U1/5'剪接位点相互作用的稳定性的贡献将被量化（特定目标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）。酵母还包含可以促进剪接体组装的因素，并且盖结合蛋白促进减数分裂调节前MRNA的剪接的机制将是用单分子方法阐明（特定目标2）。最后，通过 U2 SNFRNP需要Dead-Box ATPase PRP5的ATP水解。将使用单个分子 - ^方法阐明prp5/u2/pre-mRNA相互作用，通过动力学校对来促进分支的保真度（特定目标3）。