Structure/Function Analysis of Spliceosomal ATpases

描述（由申请人提供）：真核生物的初级转录本通常包含干预序列，必须切除以产生功能性信使rna。因此，核前mrna剪接是调节基因表达的重要步骤。选择性剪接事件在高等真核生物中产生广泛的遗传多样性并决定正常细胞发育中发挥作用。剪接模式的破坏通常与疾病有关。虽然在定义剪接的一般特征以及识别特定成分方面已经取得了很大进展，但理解这一过程的调控将需要在分子水平上对剪接机制进行分析。阐明RNA-RNA和rna -蛋白质的相互作用以及剪接体中构象变化的实现方式是这一理解的核心。对这些问题的深入了解可以通过研究atp酶的分子相互作用来获得，例如已知在剪接的特定步骤中起作用的Prp16， Prp22和Prp43。本研究提出了实验来描述剪接体DEAH-box蛋白利用ATP水解促进剪接反应的最后一步，从而形成成熟RNA，并随后从剪接体释放反应产物的机制。主要研究者建议通过重组纯化组分的部分反应来剖析剪接途径。选择酵母作为实验系统，可以将遗传学和生物化学方法强有力地结合起来，研究复杂过程中的分子相互作用。鉴于酵母和哺乳动物在基本剪接装置的结构和功能上具有很大程度的进化保守性，本研究将广泛适用于高等真核生物的pre-mRNA剪接。此外，DExH/D-box蛋白在所有主要的核酸交易中都起着重要作用，对剪接体DExH-box蛋白的分析将有可能深入了解其他家族成员的机制和功能。

DESCRIPTION (provided by applicant): Primary transcripts in eukaryotes often contain intervening sequences, which must be excised to generate functional messenger RNAs. Nuclear pre-mRNA splicing is thus an essential step in regulating gene expression. Alternative splicing events play a role in generating a broad spectrum of genetic diversity in higher eukaryotes and determine normal cell development. Disruption of splicing patterns is often associated with disease. Although much progress has been made in defining the general features of splicing as well as identifying specific components, understanding the regulation of this process will require the analysis of the splicing machinery on the molecular level. Elucidation of RNA-RNA and RNA-protein interactions and the way in which conformational changes are achieved in the spliceosome is central to this understanding. Insights into these questions can be gained by studying the molecular interactions of ATPases, such as Prp16, Prp22 and Prp43 known to function at specific steps of splicing. This proposal presents experiments to delineate the mechanisms by which spliceosomal DEAH-box proteins use ATP hydrolysis to promote the final step of the splicing reaction leading to the formation of mature RNA and of the ensuing steps to release the products of the reaction from the spliceosome. The principal investigator proposes to dissect the splicing pathway by reconstituting the partial reactions from purified components. The choice of yeast as the experimental system permits the powerful combination of genetic and biochemical approaches in studying the molecular interactions in complex processes. Given the large degree of evolutionary conservation between yeast and mammals in the structure and function of the basic splicing apparatus, the proposed studies will be broadly relevant to pre-mRNA splicing in higher eukaryotes. Moreover, DExH/D-box proteins play important roles in all major nucleic acid transactions, and the proposed analysis of the spliceosomal DExH-box proteins will likely give insight into the mechanisms and functions of other family members.