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。出乎意料的是，已经发现非编码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。本研究的长期目标是了解哺乳动物ncRNA如何在生物学重要和医学相关的实验系统中直接控制Pol II转录。这些研究将有助于辨别细胞应激期间转录是如何调节的，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。此外，我们的研究将把转录调控和功能性ncRNA领域推向新的方向。 该提案有三个具体目标。1)使用基因组测定（例如ChIP-seq、RNA-seq）和更具靶向的测定（例如ChIP、高锰酸盐处理）的组合，确定B2 RNA如何控制全基因组Pol II占有率和暂停复合物对热休克的反应的分子特征。2)使用体外蛋白质-RNA相互作用测定、因子的细胞敲低、免疫共沉淀和ChIP来识别和研究控制B2 RNA和Alu RNA活性的因子。3)了解哺乳动物Pol II RdRP活性的机制和功能，使用RdRP测定，RNA二级结构分析，蛋白质-RNA相互作用测定，体外转录实验和靶向RNA-seq的组合。 这项工作将有助于对ncRNA如何在全球范围内调节关键生物过程的新理解。此外，通过使用作为一个模型系统的广泛的，但短暂的，在热休克反应中发生的转录抑制，我们是独一无二的准备揭示新的方面的转录控制的大分子复合物组装启动子和启动后事件。此外，我们将确定和表征一个全球性的转录激活调节器，在响应热休克的功能，通过去抑制B2和Alu RNA的活动。最后，我们的实验将是第一个将哺乳动物Pol II表征为作用于细胞RNA模板的RdRP。

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). Unexpectedly, 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 long term goal of this research is to understand how mammalian ncRNAs directly control Pol II transcription in biologically important and medically relevant experimental systems. 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. Moreover, our studies will move the fields of transcriptional regulation and functional ncRNAs in new directions. The proposal has three Specific Aims. 1) Determine how B2 RNA controls genome-wide Pol II occupancy and the molecular characteristics of paused complexes in response to heat shock using a combination of genomic assays (e.g. ChIP-seq, RNA-seq) and more targeted assays (e.g. ChIP, permanganate treatment). 2) Identify and study factors that control the activities of B2 RNA and Alu RNA using in vitro protein-RNA interaction assays, cellular knockdown of factors, co-immunoprecipitations, and ChIP. 3) Understand the mechanism and functions of the mammalian Pol II RdRP activity using a combination of RdRP assays, RNA secondary structure analysis, protein-RNA interaction assays, in vitro transcription experiments, and targeted RNA-seq. Together this work will contribute a new understanding of how ncRNAs can globally regulate a critical biological process. Moreover, by using as a model system the widespread, yet transient, transcriptional repression that occurs in response to heat shock, we are uniquely poised to uncover new aspects of transcriptional control of macromolecular complex assembly on promoters and post-initiation events. In addition, we will identify and characterize a global regulator of transcriptional activation in response to heat shock that functions by derepressing the activities of B2 and Alu RNAs. Lastly, our experiments will be the first to characterize mammalian Pol II as an RdRP that acts on cellular RNA templates.