Regulation of RNA polymerase II by small RNAs

描述（由申请人提供）：所有细胞都对诸如热休克、紫外线照射和病毒感染等应激作出反应，并且部分通过改变基因表达来实现。真核细胞中调控基因表达的关键控制点是RNA聚合酶II的mRNA转录水平。在打破常规的情况下，本文讨论的初步数据确定了一种天然RNA，小鼠B2 RNA，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细胞应激反应中的作用，包括热休克、DNA损伤和病毒感染。这些研究的完成将提供对应激条件下转录是如何调节的见解，这对于理解正常细胞生长以及与疾病和有害环境条件相关的畸变至关重要。

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.