Protein RNA Rearrangements in the Spliceosome

描述(申请人提供)：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。这一过程受到高度调控，与基因转录和其他加工事件，如多聚腺苷酸化和核苷酸修饰密切相关。更好地理解前mRNA剪接对于进一步了解调控剪接的机制、控制选择性剪接的模式以及促进发育、肿瘤发生和逆转录病毒感染是至关重要的。 剪接体识别化学事件的确切位置以及如何催化反应的机制尚不清楚。该项目的长期目标是了解剪接体组件和作为催化反应底物的RNA配体之间的相互作用和重排。充分的证据表明，分支机构存在多种因素的重新安排和多重认知事件。对这些事件的研究--这些事件不是机械性的--将阐明核心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。本提案首先关注剪接体组装的第一个依赖于ATP的步骤--U2 SnRNP在分支区周围的稳定结合，并研究了ATPase Prp5的作用。作为第一个依赖于ATP的事件，这一步骤为研究剪接体ATPase的作用提供了一个独特的简化系统。 进一步的实验将集中在最近发现的由Prp5介导的U1和U2 SnRNPs之间的桥接相互作用的机制和后果。最后，使用新的遗传筛选，我们正在研究剪接体的分支位置(以及5‘和3’剪接点)和对第二催化反应至关重要的组件之间的相互作用，所识别的组件与RNA底物之间的相互作用，以及所识别的组件与剪接体的其他组件的相互作用--其中特别倾向于这些组件相互作用的机制，以帮助第二化学步骤中的反应物并列。

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.