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，这些序列必须被精确切除才能产生功能信使RNA。因此，核前mRNA剪接是调控每个真核细胞中基因表达的重要步骤。调控和选择性剪接事件在决定细胞正常发育和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，剪接模式的中断与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。尽管在确定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节需要在分子水平上分析剪接机制。阐明RNA-RNA和RNA-蛋白质的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。对这些问题的洞察可以通过研究ATPase的分子相互作用来获得，例如Prp16，已知在剪接的特定步骤发挥作用。这一建议提供了实验来描述Prp16使用ATP水解来促进剪接反应的最后一步导致成熟RNA形成的机制。我们建议通过重组纯化组分的部分反应来剖析第二步。选择酵母菌作为实验系统，可以将遗传和生化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在基本剪接装置的结构和功能上在进化上的很大程度保守，我们的研究将广泛涉及高等真核生物中的前mRNA剪接。

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.