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。出乎意料的是，已经发现了非编码RNA分子(NcRNAs)来调节mRNA的转录。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。这项研究的长期目标是了解哺乳动物ncRNAs如何在具有生物重要性和医学相关的实验系统中直接控制POL II转录。这些研究将有助于弄清细胞应激期间转录是如何调控的，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ncRNAs领域推向新的方向。该提案有三个具体目标。1)结合基因组分析(如CHIP-SEQ、RNA-SEQ)和更有针对性的分析(如CHIP、高锰酸盐处理)，确定B2 RNA如何控制全基因组POL II的占有率以及热休克反应中暂停的复合体的分子特征。2)利用体外蛋白质-RNA相互作用分析、细胞因子敲除、免疫共沉淀和芯片等方法，鉴定和研究控制B2RNA和Alu RNA活性的因素。3)通过RdRP分析、RNA二级结构分析、蛋白质-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ncRNAs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，通过使用热休克反应中广泛但短暂的转录抑制作为模型系统，我们独特地准备好揭示大分子复合体组装在启动子和启动后事件上转录控制的新方面。此外，我们将识别和表征一个全球转录激活调节因子，它通过取消抑制B2和Alu RNA的活性来响应热休克。最后，我们的实验将首次将哺乳动物Pol II描述为作用于细胞RNA模板的RdRP。 与公共卫生相关：适当控制基因表达对于维持生命和避免许多疾病和癌症至关重要；RNA聚合酶II的mRNA转录是这一过程的核心。这项拟议的研究将定义一种新的方法，通过这种方法在细胞应激期间控制信使核糖核酸转录程序。获得这一知识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。

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. PUBLIC HEALTH RELEVANCE: Properly controlling gene expression is essential to sustaining life and avoiding many diseases and cancers; mRNA transcription by RNA polymerase II is central to this process. The proposed studies will define a new means by which programs of mRNA transcription are controlled during cellular stress. Acquiring this knowledge is critical for understanding abnormalities in gene expression associated with diseases and deleterious environmental states.