STRUCTURE/FUNCTION ANALYSIS OF SPLICEOSOMAL ATPASES

高等真核生物中的初级转录本含有插入序列，必须精确切除这些插入序列才能生成功能性信使 RNA。因此，核前 mRNA 剪接是调节每个真核细胞基因表达的重要步骤。调控和选择性剪接事件在决定正常细胞发育并在高等真核生物中产生广泛的遗传多样性方面发挥着作用。 异常剪接与某些疾病有关；例如，剪接模式的破坏与 c-Hras 的致癌激活有关，c-Hras 是人类癌症中最常见的突变基因之一。尽管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，但了解该过程的调节需要在分子水平上分析剪接机制。 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一理解的核心。 通过研究 ATP 酶（例如 Prp16，已知在特定剪接步骤中发挥作用）的分子相互作用，可以深入了解这些问题。该提案提出了实验来描述 Prp16 使用 ATP 水解来促进剪接反应的最后一步，从而形成成熟 RNA 的机制。我们建议通过从纯化的组分中重构部分反应来剖析第二步。选择酵母作为实验系统，可以将遗传和生化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之间在基本剪接装置的结构和功能方面存在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体 mRNA 剪接广泛相关。

Primary transcripts in higher eukaryotes contain intervening sequences which must be precisely excised to generate functional messenger RNAs. Nuclear pre-mRNA splicing is thus an essential step in regulating gene expression in every eukaryotic cell. Regulated and alternative splicing events play a role in determining normal cell development and generate a broad spectrum of genetic diversity in higher eukaryotes. Aberrant splicing is associated with certain diseases; for example, disruption of splicing patterns has been implicated in the oncogenic activation of c-Hras, one of the most commonly mutated genes in human cancer. 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, known to function at specific steps of splicing. This proposal presents experiments to delineate the mechanisms by which Prp16 uses ATP hydrolysis to promote the final step of the splicing reaction leading to the formation of mature RNA. We propose to dissect the second step 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.