Regulation of RNA Polymerase II by Non-coding RNAs

描述(申请人提供)：在真核细胞中调节基因表达的一个关键控制点是在RNA聚合酶II(POL II)的mRNA转录过程中。非编码RNA分子(NcRNAs)作为一类新的转录调控因子出现在多种细胞系统中。小鼠B2RNA和人Alu RNA就是两个这样的ncRNAs；它们通过直接与热休克反应中的Pol II结合来抑制mRNA转录，Pol II是一种广泛用于研究细胞应激反应的模型系统。由父母拨款资助的研究的长期目标是了解与POL II结合的哺乳动物ncRNAs如何在具有生物重要性和医学相关的实验系统中控制转录。拟议的工作将通过识别和研究细胞中新的ncRNA/Pol II复合体来补充和扩展父赠款的目标。这样做将检验这样的假设，即Pol II包含一个与ncRNAs高亲和力的对接位点，不同的ncRNAs结合这个位点，并在不同的细胞系统中作为Pol II转录的反式调节因子发挥作用。该提案有一个明确的目标：鉴定和表征在不同细胞条件下形成的新型ncRNA/POL II复合体。通过免疫沉淀Pol II并对相关RNA进行测序，将发现含有ncRNAs和Pol II的新的大分子复合物。将使用两个细胞系统，一个专注于分化程序，另一个专注于细胞周期进程。对于选定的ncRNA，将通过实验确定每个ncRNA与PolII结合的百分比，以及在不同条件和细胞类型下这一比例如何变化。此外，选定的ncRNA对两种细胞类型的分化和细胞周期进程的整体程序的贡献将被调查。这些研究将把转录调控和功能ncRNA领域推向新的方向。将定义一类新的大分子复合体，它们有可能通过新的机制控制基因的表达。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复杂的ncRNAs如何在细胞分化和细胞周期进展过程中控制转录活动。

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.