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。了解这一调控过程对于理解CTD功能的错误调控如何导致导致癌症的不适当生长具有重要意义。CTD及其结合蛋白调控的转录终止机制也可能参与神经退行性疾病的发病机制。目前的建议旨在了解CTD结合蛋白Nrd1及其相关蛋白在促进转录终止方面的作用。这一目标将通过体外生化方法实现。我们将建立体外终止试验，并评估不同成分及其转基因衍生品的功能。在酿酒酵母的生长过程中终止过程是如何调节的，以及这种调节的目标是什么转录物，也将被研究。对于这个项目的这一部分，我们将采用酵母遗传学和基因组学方法的组合。特别是，我们将跟踪Nrd1和RNA聚合酶II在不同生长阶段和不同突变背景下在基因组上的分布。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，并检验该蛋白调节人类细胞转录终止的假设。这一目标将通过在RNA上定位Rbm16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与与神经退行性疾病有关的蛋白质相互作用的假设。这些实验解决了基因控制的一个最基本的方面。当细胞在生长和静止之间转换时，打开或关闭基因对于维持组织中的细胞活力至关重要。失去生长控制可能会导致癌症或细胞死亡，具体取决于细胞的类型。这项提案针对的是人们知之甚少的基因调控步骤，这些步骤是癌症和神经退行性变等疾病的基础。对这些监管过程的更坚定的理解将有助于制定治疗或预防一些人类疾病的战略。

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.