Regulation of RNA Polymerase II by Small RNAs

描述（由申请人提供）：细胞通过改变基因表达部分地响应应激。在真核细胞中调节基因表达的关键控制点是在RNA聚合酶II（Pol II）的mRNA转录期间。近年来，非编码RNA分子（ncRNA）被发现可以调节mRNA的转录。小鼠B2 RNA和人Alu RNA是两种这样的ncRNA;它们通过响应于热休克直接结合Pol II而作为mRNA转录的阻遏物发挥作用，Pol II是用于研究细胞应激反应的广泛使用的模型系统。拟议的研究将调查B2 RNA和Alu RNA如何定义转录程序，这些转录程序发生在细胞响应热休克，然后从热休克中恢复，这些ncRNA如何响应热休克进行调节，以及它们的结构和序列决定因素，允许结合Pol II和转录抑制。该提案的具体目标是：1）确定当细胞响应于热休克并从热休克中恢复时在受抑制基因处发生的分子事件，2）了解参与形成Pol II/ncRNA复合物的ncRNA和蛋白质组分，3）了解结合Pol II的ncRNA的转录抑制机制，和4）鉴定在这些ncRNA存在下去抑制转录的因子。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Pol II转录系统和核提取物。基于细胞的实验利用诸如染色质免疫沉淀和该技术的新变体、反义技术和微阵列等方法。拟议的实验将可能揭示蛋白质调节剂未观察到的转录调节的新机制和尚未鉴定的ncRNA对Pol II的调节的广泛潜力，以及产生对小鼠和人类细胞中热休克反应的转录抑制的第一个全面观点。与公共卫生的相关性：控制基因表达对生长、发育和持续生命至关重要。转录的适当调节（从DNA中产生RNA）对于维持细胞生长和分化的正常途径至关重要，从而避免在肿瘤中观察到的猖獗的细胞增殖。这些研究的完成将有助于辨别转录是如何在细胞应激过程中调节的，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.