Protein RNA Rearrangements in the Spliceosome

剪接体中蛋白质 RNA 重排

基本信息

批准号：
7259452
负责人：
CHARLES C QUERY
金额：
$ 38.71万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2008-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7259452
关键词：
3&apos Splice Site ATP phosphohydrolase ATPase Domain Active Sites Affinity Alleles Alternative Splicing Binding Biochemical Catalytic Domain Chemicals Class Code Complement Complex DDX16 Gene DNA Sequence Rearrangement Decompression Sickness Development Event Excision Exons Family Figs - dietary Fission Yeast Gene Expression Genetic Genetic Screening Genetic Transcription Goals Hela Cells Heterogeneous Nuclear RNA Human Individual Infection Introns Investigation Ligands Link Mediating Modification Molecular Conformation Mutate Mutation Nucleotides Pathway interactions Pattern Polyadenylation Process Proteins RNA RNA Splicing Reaction Relative (related person)Research Personnel Role Saccharomyces cerevisiae Scheme Screening procedure Site Spliceosome Assembly Pathway Spliceosomes Substrate Interaction System U2 Small Nuclear Ribonucleoprotein U6 small nuclear RNA Yeasts comparative falls helicase improved in vivo mRNA Precursor mutant programs research study tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. A better understanding of pre-mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how he reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events -- which are not understood mechanistically -- will elucidate interactions and rearrangements among core components and may serve as a paradigm for other rearrangements and multiple recognition events that occur elsewhere in the spliceosome. This proposal focuses first on the first ATP-dependent step of spliceosome assembly - the stable binding of U2 snRNP around the branch region, and investigates the action of an ATPase, Prp5. As the first ATP-dependent event, this step provides a uniquely simplified system, for studying the action of a spliceosomal ATPase. Further experiments will focus on the mechanism and the consequences of a recently identified bridging interaction between U1 and U2 snRNPs that is mediated by Prp5. Finally, using a new genetic screen, we are investigating interactions between the branch site (and the 5' and 3' splices sites) and components of the spliceosome critical for the second catalytic reaction, interactions between the identified components and the RNA substrate, and interactions of the identified components with other constituents of the spliceosome -- with a particular bent as to mechanism by which these components interact to help juxtapose the reactants for the second chemical step.

描述（由申请人提供）：通过剪接体从前体信使RNA中切除内含子是几乎所有人类基因表达的关键步骤。该过程受到高度调控，与基因转录和其他加工事件（例如多腺苷酸化和核苷酸修饰）紧密相关。更好地理解前 mRNA 剪接对于进一步了解调节剪接、控制选择性剪接模式以及促进发育、肿瘤发生和逆转录病毒感染的机制至关重要。剪接体识别化学事件的确切位点的机制以及反应如何被催化尚不清楚。该项目的长期目标是了解剪接体成分和作为催化反应底物的 RNA 配体之间的相互作用和重排。充足的证据表明分支位点存在多种因子重排和多种识别事件。对这些事件的研究——这些事件在机制上尚未被理解——将阐明核心组件之间的相互作用和重排，并可能作为剪接体其他地方发生的其他重排和多重识别事件的范例。该提案首先关注剪接体组装的第一个 ATP 依赖性步骤 - U2 snRNP 在分支区域周围的稳定结合，并研究 ATP 酶 Prp5 的作用。作为第一个 ATP 依赖性事件，此步骤提供了一个独特的简化系统，用于研究剪接体 ATP 酶的作用。进一步的实验将集中于最近发现的由 Prp5 介导的 U1 和 U2 snRNP 之间桥接相互作用的机制和后果。最后，使用新的遗传筛选，我们正在研究分支位点（以及 5' 和 3' 剪接位点）与对第二次催化反应至关重要的剪接体成分之间的相互作用、已识别成分与 RNA 底物之间的相互作用，以及已识别的成分与剪接体的其他成分的相互作用——特别倾向于这些成分相互作用的机制，以帮助并置第二个化学步骤的反应物。