STRUCTURE-FUNCTION ANALYSIS OF A SPLICEOSOMAL ATPASE

高等真核生物中的初级转录本通常含有介导性的 必须精确切除才能产生功能信使的序列 RNA。因此，核前-mRNA剪接是调控 基因在每个真核细胞中的表达。受监管的和替代的 剪接在决定正常细胞发育和生成 高等真核生物的广泛遗传多样性。反常的 剪接与某些疾病有关；例如，中断 剪接模式的变化与致癌活性有关 C-HRAs是人类癌症中最常见的突变基因之一。虽然 在定义剪接的一般特征方面已经取得了很大的进展 以及识别特定组件，了解法规 这一过程将需要对剪接机制进行分析 分子水平。RNA-RNA和RNA-蛋白质相互作用的研究进展 以及实现构象变化的方式 剪接体是这一理解的核心。 对这些问题的洞察可以通过研究分子 ATPase的相互作用，如PRP16，已知在特定的 拼接的步骤。这项提案提出了实验，以描绘出 蛋白质因子PRP16利用ATP水解酶的分子机制 以促进剪接反应的最后步骤，从而导致 形成成熟的RNA。PRP16蛋白的功能结构域 将通过突变分析来确定。这些研究很重要 通过将剪接反应冻结在以下温度来确定PRP16循环 不同品脱(例如，ATP结合、水解、结合或释放 剪接体)。被证明在某些功能上有缺陷的突变体 将为后续的遗传和生化研究提供有价值的工具 研究，目的是分离新的剪接因子。酵母的选择 作为一个系统，允许通用和生化的强大组合 剪接中分子相互作用的研究方法。因为 剪接过程是进化保守的，在酵母中获得了洞察力 将对高等生物的基因表达产生重要影响 真核生物。 关于PRP16功能机制的研究特别有意义 之所以感兴趣，是因为这种蛋白质似乎是一类 相关因素，Deah Box家族的成员。这些与以下内容相关 死亡(天冬氨酸-谷氨酸-丙氨酸-天冬氨酸)盒蛋白的超家族，一些成员 它们分别是依赖于RNA的ATPase和RNA解旋酶。它是 一种有趣的可能性是，每个剪接体Deah蛋白， 与PRP16一样，它使用ATP水解来促进 剪接体在剪接过程中的一个明显的步骤。因此，对 PRP16应该为理解对方的作用提供有价值的提示 家庭成员。

Primary transcripts in higher eukaryotes often contain intervening sequences which must be precisely excise 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 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;f or examples, disruption of splicing patterns has been implicated in the oncogenic activation of c-Hras, one of the most commonly mutated genes inhuman 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 of 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 molecular mechanism by which the protein factor PRP16 uses ATP hydrolysis to promote the final steps of the splicing reaction leading to the formation of mature RNA. The functional domains of the PRP16 protein will be determined by mutational analysis. These studies are important to determine the PRP16 cycle in the splicing reaction by freezing it at distinct pints (e.g. ATP-binding, hydrolysis, binding to or release from the spliceosome). Mutants that prove to be defective in certain functions will provide valuable tools for subsequent genetic and biochemical studies, aimed at isolating novel splicing factors. The choice of yeast as a system permits the powerful combination of generic and biochemical approaches in studying the molecular interactions in splicing. Because the splicing process is evolutionary conserved, insights gained in yeast will have important implications for gene expression in higher eukaryotes. Studies concerning the mechanism of PRP16 function are of particular interest because this protein appears to be a paradigm for class of related factors, members of the DEAH box family. These are related to the superfamily of DEAD (asp-glu-ala-asp) box proteins, some members of which are demonstrated RNA-dependent ATPases and RNA helicases. It is a fascinating possibility that each of the spliceosomal DEAH proteins, like PRP16, uses ATP hydrolysis to promote a conformational change in the spliceosome at a distinct step in splicing. Thus, investigations of PRP16 should offer valuable hints for understanding the role of the other members of the family.