STRUCTURE/FUNCTION ANALYSIS OF SPLICEOSOMAL ATPASES

高等真核生物中的初级转录物含有间插序列，必须精确切除以产生功能性信使RNA。因此，核前体mRNA剪接是调节每个真核细胞中基因表达的重要步骤。在高等真核生物中，受调控的剪接和选择性剪接事件在决定正常细胞发育中起作用，并产生广泛的遗传多样性。 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。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。 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Prp 16，已知其在剪接的特定步骤中起作用。该提案提出了实验来描绘Prp 16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。我们建议通过从纯化组分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。

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.