PROTEIN RNA REARRANGEMENTS IN THE SPLICEOSOME

剪接体从前体信使RNA中剔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反应如何被催化的机制尚不清楚。这个项目的长期目标是了解活性中心成分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。对这些事件的研究将阐明活性部位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。这一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，该方法将允许仅在剪接催化发生后才能识别专门接触RNA底物的分子。因此，这种方法将允许对剪接体的核心和以前未探索的催化步骤I和II的构象之间的过渡区域进行新的观察，这在以前是不可能的。此外，这使得研究毫无疑问活跃的剪接体之间的相互作用成为可能。进一步的实验将集中在已鉴定的成分与RNA底物之间的相互作用，以及已鉴定的成分与剪接体的其他成分的相互作用--以及这些成分相互作用的机制，以帮助在第一个化学步骤中并列反应物。最后，我们还将研究非锥形分支站点的识别，这些分支站点显著地出现在一些病毒和一些细胞消息中。

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.