RNA-binding RNA Polymerase II CTD-associated Proteins

描述（由申请人提供）：本提案的长期目标是了解RNA聚合酶II C末端重复结构域（CTD）在细胞生长和静止期间转录调控中的作用。了解这一调节过程对于了解CTD功能的错误调节如何导致不适当的生长从而导致癌症非常重要。CTD及其结合蛋白控制的转录终止机制也可能参与神经退行性疾病的发病机制。目前的建议是针对了解CTD结合蛋白Nrd1和相关蛋白在促进转录终止的作用。这一目标将通过体外生物化学方法实现。我们将建立一个体外终止试验，并评估不同成分及其转基因衍生物的功能。 如何终止过程中的酵母酿酒酵母的生长过程中进行调节，什么成绩单是这种调节的目标也将进行调查。对于该项目的这一部分，我们将采用酵母遗传学和基因组方法的组合。特别是，我们将遵循Nrd1和RNA聚合酶II在不同生长阶段和不同突变背景中的基因组分布。这些实验的结果将使我们了解细胞如何在静止状态下准备和执行基因调控。最后，我们将确定人类Nrd1相关蛋白Rbm16的靶点，并测试该蛋白质调节人类细胞转录终止的假设。这一目标将通过绘制Rbm16在RNA上的结合位点来实现。我们还将使用蛋白质组学方法来鉴定与Rbm16相互作用的蛋白质。这种方法将检验Rbm16与神经退行性疾病中涉及的蛋白质相互作用的假设。 这些实验解决了遗传控制的一个最基本的方面。当细胞在生长和静止之间过渡时，打开或关闭基因对维持组织中细胞的活力至关重要。 生长控制的丧失可能导致癌症或细胞死亡，这取决于细胞的类型。这项提案解决了人们对癌症和神经变性等疾病的基因调控步骤知之甚少的问题。对这些调控过程的更深入了解将有助于制定治疗或预防一些人类疾病的策略。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the role of the RNA polymerase II C-terminal repeat domain (CTD) in transcription regulation during cell growth and quiescence. Understanding this regulatory process will be important in understanding how mis-regulation of CTD function contributes to inappropriate growth leading to cancer. Transcription termination mechanisms controlled by the CTD and its binding proteins may also be involved in the pathogenesis of neurodegenerative diseases. The current proposal is directed at understanding the role of the CTD-binding protein Nrd1 and associated proteins in facilitating transcription termination. This aim will be achieved through an in vitro biochemical approach. We will establish an in vitro termination assay and assess the function of different components and their genetically-modified derivatives. How the termination process is regulated during growth in the yeast Saccharomyces cerevisiae and what transcripts are targets of this regulation will also be investigated. For this part of the project we will employ a combination of yeast genetics and genomic approaches. In particular we will follow the distribution of Nrd1 and RNA polymerase II on the genome during different stages of growth and in different mutant backgrounds. The results of these experiments will allow us to understand how cells prepare for and execute gene regulation in quiescence. Finally, we will identify the targets of the human Nrd1-related protein Rbm16 and test the hypothesis that this protein regulates transcription termination in human cells. This aim will be achieved by mapping the Rbm16 binding sites on RNA. We will also use a proteomic approached to identify proteins that interact with Rbm16. This approach will test the hypothesis that Rbm16 interacts with proteins that are implicated in neurodegenerative disease. These experiments address one of the most fundamental aspects of genetic control. Turning genes on or off as cells make the transition between growth and quiescence is critical to maintaining cell viability in tissues. Loss of growth control can lead to cancer or to cell death depending on the type of cell. This proposal addresses the poorly understood gene regulatory steps that underlie diseases like cancer and neurodegeneration. A firmer understanding of these regulatory processes will aid in development of strategies to cure or prevent a number of human diseases.