Regulation of RNA Polymerase II by Small RNAs

: 细胞对压力的反应部分是通过改变基因表达。调节的关键控制点 真核细胞中的基因表达是在RNA聚合酶II（PolII）的mRNA转录期间。最近， 已发现非编码RNA分子（ncRNA）调节mRNA转录。小鼠B2 RNA 和人Alu RNA是两种这样的ncRNA;它们通过结合 直接对Pol II反应热休克，一个广泛使用的模型系统，研究细胞应激 反应本研究将探讨B2 RNA和Alu RNA如何定义转录因子， 当细胞对热休克做出反应并从热休克中恢复时，这些ncRNA是如何 调节响应热休克，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）确定发生在 当细胞对热休克作出反应并从热休克中恢复时，被抑制的基因，2）为了了解ncRNA和 参与形成Pol II/ncRNA复合物的蛋白质组分，3）为了理解 通过结合Pol II的ncRNA的转录抑制，和4）为了鉴定解除抑制的因子， 在这些ncRNA的存在下转录。具体目标是利用体外实验和细胞- 基于分析。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了染色质免疫沉淀和一种新的变体等方法 反义技术和微阵列。拟议中的实验可能会揭示 这两种新的转录调节机制没有观察到蛋白质调节和广泛的 通过尚未鉴定的ncRNA调节Pol II的潜力，以及产生第一个全面的 在小鼠和人类细胞中对热休克反应的转录抑制的观点。 与公共卫生的相关性：控制基因表达对生长、发育和 持续的生命。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，从而避免了猖獗的细胞增殖 在肿瘤中观察到。完成这些研究将有助于辨别转录是如何 在细胞应激期间调节，这对于理解基因表达异常至关重要。 与疾病和有害的环境状态有关。

: 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 (PolII). 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 repressers 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 derepress 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.