mRNA Capping Enzyme

描述(由申请人提供)：本项目的目标是了解RNA聚合酶II的C-末端结构域如何用于将转录与基因表达的几个启动后步骤相结合。这些事件包括信使核糖核酸的封顶、剪接和多聚腺苷化，以及转录延长和终止的调节。目前的数据支持一个模型，即GTD磷酸化模式在转录的不同阶段发生变化。每一种磷酸化状态都可能被一组不同的CTD相互作用蛋白识别。这允许伸长和信使核糖核酸加工因子的动态交换，每个因子都在转录周期的适当时间(S)招募。本项目中的实验将测试这一模型，并确定CTD、其各种激酶和磷酸酶以及相关的延伸和信使核糖核酸加工因子之间的物理和功能关系。提出了四个具体目标。在第一个目标中，我们将探讨转录终止的两条途径之间的决定：mRNA多聚腺苷化/鱼雷机制和snoRNA途径。将探讨蛋白质因子、RNA序列和CTD磷酸化的作用。第二个目标将继续我们的染色质免疫沉淀研究不同突变株的延伸和mRNA加工因子的交联模式。当第二个因子发生突变时，一个因子的交联模式发生变化，这表明了一种相互依赖，这将得到生化和遗传测试。具体目标3将是继续我们的工作，开发体外系统，以重现体内观察到的CTD修饰和处理事件。这些体外系统将被用来测试和扩展来自AIMS 1和2的因子相互作用模型。具体目标4将是使用质谱学技术更好地表征CTD磷酸化的模式。所提出的实验将极大地扩展我们对基因表达的各个步骤是如何整合的理解。很明显，启动后事件在许多系统中都受到调节，以调节基因的活性。清楚地了解基因表达的基本机制将为未来的治疗提供基础，包括基因替代治疗和直接调节细胞和病毒基因表达。

DESCRIPTION (provided by applicant): The goal of this project is to understand how the C-terminal domain of RNA polymerase II is used to couple transcription with several post-initiation steps in gene expression. These events include mRNA capping, splicing, and polyadenylation, as well as regulation of transcription elongation and termination. Current data supports a model in which the pattern of GTD phosphorylation changes at different stages of transcription. Each phosphorylation state may be recognized by a distinct set of CTD-interacting proteins. This allows a dynamic exchange of elongation and mRNA processing factors, each one recruited at the appropriate time(s) of the transcription cycle. The experiments in this project will test this model and identify physical and functional relationships between the CTD, its various kinases and phosphatases, and associated elongation and mRNA processing factors. Four specific aims are proposed. In the first aim, we will explore the decision between two pathways for transcription termination: the mRNA polyadenylation/torpedo mechanism and the snoRNA pathway. The role of protein factors, RNA sequences, and CTD phosphorylation will be probed. The second aim will continue our chromatin immunoprecipitation studies of the crosslinking patterns of elongation and mRNA processing factors in various mutant strains. Changes in crosslinking patterns of one factor when a second is mutated suggests an interdependence that will be tested biochemically and genetically. Specific Aim 3 will be to continue our work on developing in vitro systems for reproducing the CTD modification and processing events observed in vivo. These in vitro systems will be used to test and extend the models of factor interactions derived from Aims 1 and 2. Specific Aim 4 will be to better characterize the patterns of CTD phosphorylation using mass spectroscopic techniques. The experiments proposed will significantly extend our understanding of how various steps in gene expression are integrated. It is clear that post-initiation events are regulated in many systems for modulation of gene activity. A clear understanding of the fundamental mechanisms of gene expression will provide the groundwork for future therapies, including gene replacement therapies and direct modulation of cellular and viral gene expression.