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反应如何被催化的机制尚不清楚。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--这些事件不是从机械上理解的--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机器中重排的范例。这项建议侧重于剪接体动力学改变影响剪接保真度的机制。实验将调查ATPase Prp5对剪接体组装的保真度的贡献。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鉴定分支位点亲核体的机制上，它将在酵母中使用“正交剪接体”系统。最后，使用基因筛查，我们正在研究剪接体的组件，这些组件强烈地改变了从剪接的第一步到第二步的过渡。这导致了一种新的观点，通过改变第一步和第二步构象之间的平衡，使用非共识剪接位点的机制。这些研究将有助于阐明剪接体功能的重要特征--因为这些位于Cef1、Prp8和U6SnRNA中的新突变体具有强烈改变剪接体在突变的剪接/分支位点上功能的特征。

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.