Protein RNA Rearrangements in the Spliceosome

剪接体中蛋白质 RNA 重排

• 批准号: 6823826
• 负责人: CHARLES C QUERY
• 金额: $ 37万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 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/6823826
• 关键词: RNA binding protein; RNA splicing; catalyst; crosslink; genetic screening; intermolecular interaction; introns; laboratory rabbit; ligands; messenger RNA; molecular rearrangement; protein protein interaction; protein structure function; spliceosomes; yeast two hybrid system

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）：剪接体从前体信使RNA切除内含子是几乎所有人类基因表达的关键步骤。该过程受到高度调节，与基因和其他加工事件的转录（例如聚腺苷酸化和核苷酸修饰）的转录有联系。更好地理解MRNA剪接对于进一步了解调节剪接的机制至关重要，控制替代剪接的模式以及有助于发育，造成肿瘤发生和逆转录病毒感染。 剪接体识别化学事件的确切位点以及如何催化他的反应的机制尚不清楚。该项目的长期目标是了解剪接体成分与RNA配体之间的相互作用和重排，后者是催化反应的底物。充分的证据表明，分支机构的多个因素和多个识别事件的重新排列。对这些事件的调查 - 从机械上讲是无法理解的 - 将阐明核心组件之间的相互作用和重排，并可以作为其他重排的范式，以及剪接体中其他地方发生的多个识别事件。该建议首先关注剪接组装的第一个依赖ATP依赖性步骤 - U2 SNRNP围绕分支区域的稳定结合，并研究了ATPase，PRP5的作用。作为第一个依赖ATP的事件，此步骤提供了一个独特的简化系统，用于研究剪接ATPase的作用。 进一步的实验将集中于由PRP5介导的U1和U2 SNRNP之间最近确定的桥接相互作用的机理和后果。 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第二个化学步骤。