PROTEIN RNA REARRANGEMENTS IN THE SPLICEOSOME

剪接体中的蛋白质 RNA 重排

• 批准号: 6612637
• 负责人: CHARLES C QUERY
• 金额: $ 33.48万
• 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 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612637
• 关键词: RNA binding protein; RNA splicing; crosslink; intermolecular interaction; introns; messenger RNA; molecular rearrangement; protein protein interaction; protein structure function; spliceosomes; yeast two hybrid system

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 the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between active site 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 active site 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 identification of all the proteins interacting with one site throughout the entire spliceosome cycle, with emphasis on a new approach that will allow indentification of molecules specifically contacting the RNA substrate only after splicing catalysis has occurred. Thus, this approach will allow a new view into the core of the spliceosome and the previously unexplored areas of transitions between the conformations for catalytic steps I and II that has not previously been possible. In addition, this allows investigation of interactions within unquestionably active spliceosomes. Further experiments will focus on interactions between the identified components and the RNA substrate, and on 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 first chemical step. Finally, we will also investigate the recognition of non-conanical branch sites, which occur notably in some viral and a few cellular messages.

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催化尚不清楚。 该项目的长期目标是了解活性位点成分与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（在机制上尚未被理解）将阐明活性位点成分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。 该提案首先侧重于鉴定在整个剪接体循环中与一个位点相互作用的所有蛋白质，重点是一种新方法，该方法将允许识别仅在剪接催化发生后特异性接触RNA底物的分子。 因此，这种方法将为剪接体的核心和催化步骤 I 和 II 的构象之间以前未探索的转变区域提供新的视角，这在以前是不可能的。此外，这还可以研究毫无疑问活跃的剪接体内的相互作用。进一步的实验将集中于已识别成分与 RNA 底物之间的相互作用，以及已识别成分与剪接体其他成分之间的相互作用 - 特别倾向于这些成分相互作用的机制，以帮助并置第一种化学物质的反应物步。 最后，我们还将研究非圆锥分支位点的识别，这些分支位点尤其出现在一些病毒和一些细胞消息中。