Structure/Function Analysis of Spliceosomal ATpases

真核生物中的初级转录物通常含有插入序列，必须将其切除以产生功能性信使RNA。因此，核前体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的细胞发育中起作用。剪接模式的破坏通常与疾病有关。虽然已经取得了很大的进展，在定义剪接的一般特征，以及确定特定的组件，了解这个过程的调节将需要在分子水平上的剪接机制的分析。阐明RNA-RNA和RNA-蛋白质相互作用以及剪接体中实现构象变化的方式是这种理解的核心。 通过研究ATP酶的分子相互作用可以深入了解这些问题，例如已知在剪接的特定步骤中起作用的Prp 16，Prp 22和Prp 43。该提案提出了实验来描绘剪接体DEAH盒蛋白利用ATP水解促进剪接反应的最后一步的机制，导致成熟RNA的形成和随后的步骤，以释放剪接体的反应产物。我们建议通过从纯化组分重构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有效地结合起来，研究复杂过程中的分子相互作用。鉴于酵母和哺乳动物之间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，我们的研究将与高等真核生物中的前mRNA剪接广泛相关。此外，DExH/D-box蛋白在所有主要的核酸交易中起着重要的作用，并且对剪接体DExH-box蛋白的拟议分析可能会深入了解其他家族成员的机制和功能。性能现场=

EXCEED THE SPACE PROVIDED, 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. We propose 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, our studies will be broadly relevant to pre-mRNA splicing in higher eukaryotes. Moreover, DExH/D-box proteins play important roles in atl 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. PERFORMANCE SITE ========================================Section End===========================================