Protein RNA Rearrangements in the Spliceosome

描述(由申请人提供)： 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。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U1-U2蛋白相互作用网络和ATPase Prp5对内含子定义和剪接体组装保真度的贡献。作为第一个依赖于ATP的事件，这一步骤为剪接体组装提供了独特的承诺，并为研究剪接体ATPase的作用提供了一个简化的系统。进一步的实验将集中在第二步剪接的3‘剪接位点底物的结合部位，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相互作用在从第一步到第二步剪接的转变中的作用。总之，在这些目标中提出的模型将极大地提高我们对RNA-RNA相互作用在剪接连续阶段的动态范围的理解。

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.