Regulation of RNA polymerase ll by small RNAs

小RNA对RNA聚合酶II的调节

• 批准号: 6942999
• 负责人: James Goodrich
• 金额: $ 29.48万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-19 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6942999
• 关键词: DNA damage; DNA directed RNA polymerase; RNA; RNA binding protein; RNA interference; animal tissue; antisense nucleic acid; enzyme activity; enzyme inhibitors; gene expression; gene induction /repression; genetic regulation; genetic transcription; immunoprecipitation; messenger RNA; nuclear runoff assay; protein structure function; ribozymes; virus infection mechanism

DESCRIPTION (provided by applicant): All cells respond to stress such as heat shock, UV irradiation, and viral infection, and do so in part by altering gene expression. A critical control point for regulating gene expression in eukaryotic cells is at the level of mRNA transcription by RNA polymerase II. In a break with paradigm, the preliminary data discussed here identify a natural RNA, mouse B2 RNA, which can bind to and inhibit transcription by RNA polymerase II. The proposed studies will investigate the role of B2 RNA as a transcriptional repressor in response to cellular stresses including heat shock, DNA damage, and viral infection. Completion of these studies will provide insight into how transcription is regulated under conditions of stress, which is vitally important in understanding normal cell growth as well as aberrations associated with diseases and deleterious environmental conditions. The specific aims of the proposal are the following: 1) To study the function of B2 RNA as a negative regulator of the transcriptional response to heat shock, DNA damage, and viral infection in mouse cells. 2) To determine the molecular mechanism by which B2 RNA inhibits transcription by RNA polymerase II. 3) To map the regions of B2 RNA and RNA polymerase II that interact, and in doing so develop a small nucleic acid inhibitor of the transcriptional response to stress. 4) To characterize human RNAs that bind to RNA polymerase II and regulate transcription in response to heat shock, DNA damage, and viral infection. The specific aims utilize both in vitro experiments and cell-based assays. The in vitro experiments employ a purified RNA polymerase II transcription system as well as nuclear extracts prepared from stressed mouse cells. The cell-based experiments utilize techniques to decrease levels of regulatory RNAs in cells such as antisense, ribozyme, and RNAi, as well as techniques to monitor RNA polymerase II activity such as nuclear run-on assays and chromatin immunoprecipitations. The proposed experiments have the potential to show that natural small noncoding RNAs can regulate transcription by targeting RNA polymerase II, to experimentally demonstrate a function for the SINE encoded B2 RNA, and to propose a mechanism by which general mRNA transcription is repressed in response to cellular stress.

描述（由申请人提供）：所有细胞都对应激反应，如热休克、紫外线照射和病毒感染，部分通过改变基因表达来实现。调节真核细胞中基因表达的关键控制点是RNA聚合酶II在mRNA转录水平上的作用。打破常规，这里讨论的初步数据确定了一种天然RNA，小鼠B2 RNA，它可以结合并抑制RNA聚合酶II的转录。拟议的研究将探讨B2 RNA作为转录抑制因子在应对细胞应激（包括热休克、DNA损伤和病毒感染）中的作用。这些研究的完成将提供深入了解转录是如何调节的条件下的压力，这是至关重要的理解正常细胞生长以及与疾病和有害的环境条件相关的畸变。 具体目标如下：1）研究B2 RNA作为小鼠细胞对热休克、DNA损伤和病毒感染的转录反应的负调控因子的功能。2)确定B2 RNA抑制RNA聚合酶II转录的分子机制。3)绘制B2 RNA和RNA聚合酶II相互作用的区域，并在此过程中开发一种小的核酸抑制剂，抑制对应激的转录反应。4)表征与RNA聚合酶II结合并调节转录以响应热休克、DNA损伤和病毒感染的人RNA。 具体目标利用体外实验和基于细胞的测定。体外实验采用纯化的RNA聚合酶II转录系统以及从应激小鼠细胞制备的核提取物。基于细胞的实验利用技术来降低细胞中调控RNA的水平，如反义、核酶和RNAi，以及监测RNA聚合酶II活性的技术，如核连续测定和染色质免疫沉淀。拟议的实验有可能表明，天然小的非编码RNA可以通过靶向RNA聚合酶II调节转录，实验证明了SINE编码的B2 RNA的功能，并提出了一种机制，通过这种机制，一般的mRNA转录在细胞应激反应中受到抑制。