PROTEIN RNA REARRANGEMENTS IN THE SPLICEOSOME

通过剪接体从前体信使RNA切除内含子是几乎所有人类基因表达的关键步骤。 这一过程受到高度调控，与基因转录和其他加工事件（如聚腺苷酸化和核苷酸修饰）紧密相关。 更好地了解前mRNA剪接将是至关重要的，以进一步了解机制，调节剪接，控制模式的选择性剪接，并有助于发展，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的机制以及反应如何被催化的机制还没有很好的理解。 该项目的长期目标是了解活性位点组分与作为催化反应底物的RNA配体之间的相互作用和重排。 有充分的证据表明，在分支位点上存在多个因子的重排和多个识别事件。 这些事件的调查-这是不理解的机制-将阐明活性位点组件之间的相互作用和重排，并可能作为一个范例，其他重排和多个识别事件发生在剪接体的其他地方。 该建议首先集中在识别所有的蛋白质相互作用的一个网站在整个剪接体循环，重点是一种新的方法，将允许识别分子特异性接触RNA底物后，剪接催化已经发生。 因此，这种方法将允许一个新的观点到核心的剪接体和以前未探索的领域之间的转换的构象催化步骤I和II，以前是不可能的。此外，这使得调查的相互作用内毫无疑问积极剪接体。进一步的实验将集中在确定的成分和RNA底物之间的相互作用，以及确定的成分与剪接体的其他成分之间的相互作用-特别是关于这些成分相互作用以帮助并列反应物进行第一化学步骤的机制。 最后，我们还将研究识别的非conanical分支网站，这发生在一些病毒和一些细胞的消息显着。

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.