Protein RNA Rearrangements in the Spliceosome

描述（由申请人提供）： 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。更好地理解前mRNA剪接对于进一步理解调节剪接、控制选择性剪接模式以及促进发育、肿瘤发生和逆转录病毒感染的机制至关重要。 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。这个项目的长期目标是了解剪接体成分和RNA配体之间的相互作用和重排，RNA配体是催化反应的底物。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-将阐明核心组件之间的相互作用和重排，并可能作为一个范例的spliceosome和其他RNP机器的重排。这项建议的重点是机制，改变剪接体动态影响剪接保真度。 实验将研究U1-U2蛋白质相互作用网络和ATP酶Prp 5对内含子定义和剪接体组装保真度的贡献。作为第一个ATP依赖的事件，这一步骤提供了一个独特的承诺剪接体组装和简化的系统，研究剪接体ATP酶的行动。进一步的实验将集中在3'剪接位点底物的结合位点上，用于剪接的第二步，这将在酵母中使用“正交剪接体”系统，以及第二步底物取代催化核心中的第一步产物的机制。最后，我们正在研究新的RNA-RNA相互作用在从剪接的第一步到第二步的过渡中的作用。总之，这些目标中提出的模型将大大提高我们对连续剪接阶段RNA-RNA相互作用动态范围的理解。

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 contributions of the U1-U2 protein-interaction network and the ATPase Prp5 to intron definition and to the fidelity of spliceosome assembly. As the first ATP-dependent event, this step provides a unique commitment to spliceosome assembly and a simplified system for studying the action of a spliceosomal ATPase. Further experiments will focus on the binding site of the 3' splice site substrate for the second step of splicing, which will use an 'orthogonal spliceosome' system in yeast, and on the mechanism by which the second-step substrate replaces first-step product in the catalytic core. Finally, we are investigating the role of novel RNA-RNA interactions in the transition from the first-to-second step of splicing. Together, the models proposed in these aims will greatly improve our understanding of the dynamic range of RNA-RNA interactions at consecutive stages of splicing.