Regulation of RNA Polymerase II by Non-coding RNAs

描述（由申请人提供）：调节真核细胞中基因表达的关键控制点是通过RNA聚合酶II（Pol II）进行mRNA转录。非编码RNA分子（ncRNA）已经作为一类新的转录调节因子出现在多种细胞系统中。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。由父母资助的研究的长期目标是了解哺乳动物ncRNA如何结合到Pol II控制生物学重要和医学相关的实验系统中的转录。拟议的工作将通过鉴定和研究细胞中新的ncRNA/Pol II复合物来补充和扩展母基金的目标。这样做将测试Pol II包含ncRNA的高亲和力对接位点的假设，并且不同的ncRNA结合该位点并在不同的细胞系统中作为Pol II转录的反式调节剂发挥作用。 该提案有一个具体目标：鉴定和表征在不同细胞条件下形成的新型ncRNA/Pol II复合物。通过免疫沉淀Pol II并对相关RNA进行测序，将发现含有ncRNA和Pol II的新的大分子复合物。将使用两种细胞系统，一种侧重于分化程序，另一种侧重于细胞周期进程。对于选定的ncRNA，将通过实验确定每种ncRNA与Pol II结合的百分比，以及在不同条件和细胞类型下如何变化。此外，将研究所选的ncRNA对两种细胞类型中分化和细胞周期进程的整体程序的贡献。 这些研究将使转录调控和功能性ncRNA研究领域向新的方向发展。细胞中一类新的大分子复合物将被定义为具有通过新机制控制基因表达的潜力。拟议工作的结果将为未来的研究提供平台，这些研究将研究与Pol II复合的ncRNA如何在细胞分化和细胞周期进展过程中控制转录活性。

DESCRIPTION (provided by applicant): A critical control point for regulating gene expression in eukaryotic cells is during mRNA transcription by RNA polymerase II (Pol II). Non-coding RNA molecules (ncRNAs) have emerged as a new class of transcriptional regulators in a variety of cellular systems. 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 long term goal of research funded by the parent grant is to understand how mammalian ncRNAs that bind to Pol II control transcription in biologically important and medically relevant experimental systems. The proposed work will complement and extend the goals of the parent grant by identifying and investigating new ncRNA/Pol II complexes in cells. Doing so will test the hypothesis that Pol II contains a high-affinity docking site for ncRNAs, and that diverse ncRNAs bind this site and function as trans-regulators of Pol II transcription in different cellular systems. The proposal hs one Specific Aim: Identify and characterize novel ncRNA/Pol II complexes that form under distinct cellular conditions. New macromolecular complexes containing ncRNAs and Pol II will be found by immunoprecipitating Pol II and sequencing the associated RNAs. Two cellular systems will be used, one focusing on a program of differentiation and the other on cell cycle progression. For selected ncRNAs the percent of each ncRNA bound to Pol II, and how this changes under different conditions and cell types, will be experimentally determined. In addition, the contribution the selected ncRNAs make to the overall programs of differentiation and cell cycle progression in the two cell types will be investigated. These studies will move the fields o transcriptional regulation and functional ncRNAs in new directions. A new class of macromolecular complexes in cells will be defined that have potential to control gene expression through novel mechanisms. Results from the proposed work will provide the platform for future lines of research that will investigate how ncRNAs that complex with Pol II control transcriptional activity during programs of cellular differentiation and cell cycle progression.