PROTEIN RNA REARRANGEMENTS IN THE SPLICEOSOME

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（如聚腺苷酸化和核苷酸修饰）紧密相关。更好地了解pre-mRNA剪接对于进一步理解调节剪接的机制、控制可选剪接模式以及促进发育、肿瘤发生和逆转录病毒感染至关重要。剪接体识别化学事件的确切位点的机制以及如何催化反应尚不清楚。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。充分的证据表明，在分支部位存在多种因素重排和多种识别事件。对这些未被机械理解的事件的研究将阐明活性位点组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。该建议首先侧重于鉴定在整个剪接体周期中与一个位点相互作用的所有蛋白质，重点是一种新的方法，该方法将允许鉴定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以前未探索的催化步骤I和II的构象之间的过渡提供新的视角，这在以前是不可能的。此外，这允许在毫无疑问的活跃剪接体内的相互作用的调查。进一步的实验将集中于已识别的组分与RNA底物之间的相互作用，以及已识别的组分与剪接体的其他组分之间的相互作用——这些组分相互作用的特定机制有助于在第一个化学步骤中将反应物并列。最后，我们还将研究非圆锥分支位点的识别，这在一些病毒和一些细胞信息中明显发生。

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.