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中的几个后起始步骤偶联。这些事件包括mRNA加帽、剪接和聚腺苷酸化，以及转录延伸和终止的调节。目前的数据支持一种模型，其中GTD磷酸化的模式在转录的不同阶段发生变化。每种磷酸化状态都可以被一组不同的CTD相互作用蛋白识别。这允许延伸和mRNA加工因子的动态交换，每一个在转录周期的适当时间被募集。本项目中的实验将测试该模型，并确定CTD，其各种激酶和磷酸酶，以及相关的延伸和mRNA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，我们将探索两种转录终止途径之间的决定：mRNA聚腺苷酸化/鱼雷机制和snoRNA途径。蛋白质因子，RNA序列和CTD磷酸化的作用将被探讨。第二个目标将继续我们的染色质免疫沉淀研究的交联模式的延伸和mRNA加工因子在各种突变株。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性，这将在生物化学和遗传学上进行测试。具体目标3将是继续我们的工作，在体外系统开发再现CTD的修改和处理在体内观察到的事件。这些体外系统将用于测试和扩展源自目标1和2的因子相互作用模型。具体目标4是使用质谱技术更好地表征CTD磷酸化的模式。提出的实验将大大扩展我们对基因表达中各个步骤是如何整合的理解。很明显，在许多调节基因活性的系统中，起始后事件受到调节。对基因表达的基本机制的清楚理解将为未来的治疗提供基础，包括基因替代疗法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.