Protein RNA Rearrangements in the Spliceosome

描述（由申请人提供）：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剪接体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。对这些事件的研究将阐明核心组件之间的相互作用和重排，并可能作为剪接体和其他RNP机器重排的范例。这一建议的重点是改变剪接体动力学影响剪接保真度的机制。实验将研究atp酶Prp5对剪接体组装保真度的贡献。作为第一个atp依赖事件，这一步为研究剪接体atp酶的作用提供了一个独特的简化系统。进一步的实验将集中于在酵母中使用“正交剪接体”系统鉴定分支位点亲核试剂的机制。最后，使用遗传筛选，我们正在研究剪接体中强烈改变剪接从第一步到第二步转变的成分。这导致了通过改变第一步和第二步构象之间的平衡而使用非一致剪接位点的机制的新观点。这些研究将有助于阐明剪接体功能的重要特征，因为Cef1、Prp8和U6 snRNA的这些新突变具有强烈改变剪接体在突变剪接/分支位点上的功能的特征。

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 the 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 rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which altered spliceosomal dynamics impact splicing fidelity. Experiments will investigate the contribution of the ATPase Prp5 to the fidelity of spliceosome assembly. 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 of identification of the branch site nucleophile, which will use an 'orthogonal spliceosome' system in yeast. Finally, using a genetic screen, we are investigating components of the spliceosome that strongly alter the transition from the 1st to 2nd step of splicing. This has led to a new view of the mechanism by which non-consensus splice sites are used, through alteration of the equilibrium between 1st and 2nd step conformations. These studies will help elucidate important features of spliceosome function - as these new mutants in Cef1, Prp8, and U6 snRNA have the feature of strongly altering the function of the spliceosome on mutated splice/branch sites.