Regulation of RNA Polymerase II by Small RNAs

描述(申请人提供)：细胞对压力的反应部分是通过改变基因表达。真核细胞基因表达调控的一个关键控制点是RNA聚合酶II(Pol II)转录过程中的基因转录。最近，人们发现非编码RNA分子(NcRNAs)可以调节mRNA的转录。小鼠B2RNA和人Alu RNA就是两个这样的ncRNAs；它们通过直接与热休克反应中的Pol II结合来抑制mRNA转录，Pol II是一种广泛用于研究细胞应激反应的模型系统。拟议的研究将调查B2 RNA和Alu RNA如何定义细胞在热休克反应后恢复时发生的转录程序，这些ncRNAs是如何在热休克反应中调节的，以及它们的结构和序列决定因素，允许与Pol II结合和转录抑制。该提案的具体目的是：1)确定细胞对热休克的反应和恢复过程中抑制基因发生的分子事件；2)了解形成Pol11/ncRNA复合体的ncRNA和蛋白质组分；3)了解与PolII结合的ncRNAs的转录抑制机制；4)确定在这些ncRNAs存在的情况下抑制转录的因素。具体目的是利用体外实验和基于细胞的分析。体外实验使用了纯化的Pol II转录系统和核提取物。基于细胞的实验利用了染色质免疫沉淀和这项技术的一种新变体、反义技术和微阵列等方法。拟议的实验可能会揭示蛋白质调节器没有观察到的转录调控的新机制，以及通过尚不确定的ncRNAs调控POL II的广泛潜力，并产生第一个关于热休克反应的转录抑制的全面观点。与公众健康相关：控制基因表达对生长、发育和可持续生命至关重要。适当的转录调控(从DNA中制造RNA)对于维持正常的细胞生长和分化途径至关重要，从而避免肿瘤中观察到的疯狂的细胞增殖。这些研究的完成将有助于弄清细胞应激期间转录是如何调控的，这对于理解与疾病和有害环境状态相关的基因表达异常至关重要。

DESCRIPTION (provided by applicant): Cells respond to stress in part by altering gene expression. A critical control point for regulating gene expression in eukaryotic cells is during mRNA transcription by RNA polymerase II (Pol II). Recently, non-coding RNA molecules (ncRNAs) have been found to regulate mRNA transcription. Mouse B2 RNA and human Alu RNA are two such ncRNAs; they function as repressors of mRNA transcription by binding directly to Pol II in response to heat shock, a widely used model system for studying the cellular stress response. The proposed studies will investigate how B2 RNA and Alu RNA define the transcriptional program that occurs as cells respond to and then recover from heat shock, how these ncRNAs are regulated in response to heat shock, and their structural and sequence determinants that allow binding to Pol II and transcriptional repression. The specific aims of the proposal are: 1) To determine the molecular events that occur at repressed genes as cells respond to and recover from heat shock, 2) To understand the ncRNA and protein components involved in forming Pol ll/ncRNA complexes, 3) To understand the mechanism of transcriptional repression by ncRNAs that bind Pol II, and 4) To identify factors that de-repress transcription in the presence of these ncRNAs. The specific aims utilize in vitro experiments and cell-based assays. The in vitro experiments employ a purified Pol II transcription system and nuclear extracts. The cell-based experiments utilize methods such as chromatin immunoprecipitations and a novel variation of this technique, antisense technologies, and microarrays. The proposed experiments will likely reveal both novel mechanisms of transcriptional regulation not observed with protein regulators and the broad potential for regulation of Pol II by yet unidentified ncRNAs, as well as generate the first comprehensive view of transcriptional repression in response to heat shock in both mouse and human cells. Relevance to public health: Controlling gene expression is essential to growth, development, and sustained life. The proper regulation of transcription (making RNA from DNA) is essential to maintaining normal pathways of cell growth and differentiation, thereby avoiding the rampant cell proliferation observed in tumors. Completion of these studies will contribute to discerning how transcription is regulated during cellular stress, which is critical for understanding abnormalities in gene expression associated with diseases and deleterious environmental states.