Regulation of RNA Polymerase II by Non-coding RNAs

描述（由申请人提供）：在真核细胞中调节基因表达的关键控制点是RNA聚合酶II （Pol II）转录mRNA的过程。出乎意料的是，非编码RNA分子（ncRNAs）被发现调节mRNA转录。小鼠B2 RNA和人Alu RNA就是这两种ncRNAs；热休克是研究细胞应激反应的一个广泛使用的模型系统，它们通过直接结合Pol II来抑制mRNA转录。这项研究的长期目标是了解哺乳动物ncRNAs如何在生物学和医学相关的实验系统中直接控制Pol II转录。这些研究将有助于识别细胞应激过程中转录是如何被调节的，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，我们的研究将推动转录调控和功能性ncrna领域向新的方向发展。该提案有三个具体目标：1)利用基因组分析（如ChIP-seq， RNA-seq）和更有针对性的分析（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占据和暂停复合物在热休克反应中的分子特征。2)利用体外蛋白-RNA相互作用、细胞敲低因子、共免疫沉淀、ChIP等方法，鉴定和研究B2 RNA和Alu RNA活性的控制因子。3)通过RdRP分析、RNA二级结构分析、蛋白-RNA相互作用分析、体外转录实验和靶向RNA-seq等方法，了解哺乳动物Pol II RdRP活性的机制和功能。总之，这项工作将有助于对ncrna如何在全球范围内调节一个关键的生物过程有一个新的理解。此外，通过使用广泛而短暂的转录抑制作为模型系统来响应热休克，我们独特地揭示了启动子和起始后事件上大分子复合物组装的转录控制的新方面。此外，我们将确定并描述一个响应热休克的转录激活的全局调节因子，该调节因子通过抑制B2和Alu rna的活性发挥作用。最后，我们的实验将首次将哺乳动物Pol II定性为作用于细胞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.