Elucidation of the Histone 3' End Formation Mechanism

描述（由申请人提供）： 这个提议的广泛的长期目标是确定参与组蛋白mRNA 3'端形成的新因子，包括生物化学和遗传学，并在体内和体外表征这些蛋白质。Specific Aim 1旨在使用经典和创新生物化学技术的组合来鉴定和纯化以功能相关方式与HeLa细胞核提取物中的组蛋白前mRNA结合的蛋白质。具体目标2将涉及使用一种新的基于GFP的遗传筛选在果蝇中进行，旨在鉴定该过程中的功能缺失突变体。该筛选将在胚胎分选机的帮助下进行至接近饱和水平。具体目标3是开发一种体内系统，以研究已鉴定因子的功能相关性并对其进行表征。该体内测定也可应用于哺乳动物细胞中基于siRNA的新型遗传筛选。还将开发一种体外系统来研究在活性转录的背景下组蛋白mRNA 3'末端的形成。组蛋白mRNA 3'末端的形成与细胞周期和DNA复制密切相关，因此了解这一过程可能会导致更有效的化疗药物的产生。

DESCRIPTION (provided by applicant): The broad long-term objectives to this proposal are to identify new factors involved in histone mRNA 3' end formation both biochemically and genetically and to characterize those proteins in vivo and in vitro. Specific Aim 1 is designed to use a combination of classical and innovative biochemical techniques to identify and purify proteins binding in a functionally relevant manner to histone pre-mRNA in HeLa nuclear extract. Specific Aim 2 will involve the use of a novel GFP based genetic screen to be performed in Drosophila melanogaster aimed at identifying loss of function mutants in the process. This screen will be performed to near saturating levels with the aid of an embryo sorter. Specific Aim 3 is to develop an in vivo system to study the function relevance of identified factors as well as to characterize them. This in vivo assay may also be applied in a novel siRNA-based genetic screen in mammalian cells. An in vitro system will also be developed to study histone mRNA 3' end formation in the context of active transcription. Histone mRNA 3' end formation is intimately related to the cell cycle and DNA replication, thus understanding this process may lead to the production of more effective chemotherapeutic agents.