Molecular Switches: Regulatory RNAs Targeting Nuclear Hormone Receptor Promoters

描述（由申请人提供）：转录组分析的最新结果表明，大部分基因组被转录，RNA转录物由两条DNA链产生。这些非编码RNA转录本在哺乳动物细胞中的生物学意义尚不清楚。我们认为非编码RNA转录本与小的非编码反基因RNA和蛋白质在基因启动子处相互作用以调节表达。启动子非编码RNA之间的关联控制基因表达可能代表了一种新的调控机制。目的：我们的主要目标（目的I）是通过鉴定相关蛋白质，并确定它们如何与启动子DNA和RNA转录物相互作用，进一步阐明反基因RNA激活或抑制PR基因表达的机制。将进行基于细胞的测定以鉴定所涉及的蛋白质。为了理解蛋白质-核酸相互作用，我们将使用设计的方法分离结合到PR基因内的启动子DNA或PR基因产生的RNA转录物的特定蛋白质。这些数据将使我们能够建立一个机制模型，RNA介导的控制基因表达的基因启动子。我们的第二个目标是测试激活agRNA是否通过诱导基因转录、增强进行性延伸和/或稳定RNA转录物来增加基因表达。检测agRNA介导的对这些机制的影响将有助于理解agRNA作用机制的关键方面。我们的第三个第二个目标（目的III）是调查存在的内源性抗基因RNA调节PR基因。为了识别潜在的反基因RNA，我们将使用生物信息学方法，筛选microRNA文库中与PR基因启动子内位点互补的序列。我们将使用生物化学和基于细胞的测定来测试在我们的筛选中鉴定的RNA“命中”的参与。重要性：反义RNA转录本和启动子上的小的非编码RNA之间的关联来控制基因表达可能代表了一种新的调控机制。了解其机制将扩展我们对为什么大多数基因组被转录但不翻译成蛋白质以及基因表达如何由启动子处的RNA调节的理解。该提案将通过证明RNA可用于靶向基因启动子并激活基因表达来影响研究。拟议的实验旨在提供数据，这些数据通常对研究人员在培养细胞和动物模型中操纵其他基因有用。公共卫生相关性：了解非编码RNA如何选择性地激活或抑制基因启动子的基因表达，可能会揭示以前未被认识到的基因调控的一般机制。许多疾病状态是由于一个关键蛋白质的表达减少。探索RNA特异性增加靶基因表达的能力将鼓励疾病治疗和开发治疗线索的新方法。

DESCRIPTION (provided by applicant): Recent results from transcriptome analysis demonstrate that most of the genome is transcribed and that RNA transcripts are generated from both DNA strands. The biological significance of these non-coding RNA transcripts in mammalian cells remains unclear. We propose that non-coding RNA transcripts interact with small non-coding antigene RNAs and proteins at gene promoters to regulate expression. Association between non-coding RNAs at promoters to control gene expression may represent a novel regulatory mechanism. Objectives: Our primary goal (Aim I) is to further delineate the mechanism by which antigene RNAs activate or repress PR gene expression by identifying the proteins involved, and determining how they interact with promoter DNA and RNA transcripts. Cell-based assays will be performed to identify the proteins involved. To understand protein-nucleic acid interactions we will use methods designed to isolate specific proteins bound to promoter DNA within the PR gene or RNA transcripts that are generated from the PR gene. These data will allow us to build a mechanistic model for RNA-mediated control of gene expression at gene promoters. Our second goal is to test whether activating agRNAs increase gene expression by inducing gene transcription, enhancing processive elongation and/or stabilizing RNA transcripts. Testing for agRNA-mediated effects on these mechanisms will aid in understanding key aspects of the agRNA mechanism of action. Our third second goal (Aim III) is to investigate the existence of endogenous antigene RNAs that regulate the PR gene. To identify potential antigene RNAs we will use bioinformatics approaches that screen microRNA libraries for sequences complementary to sites within the PR gene promoter. We will use biochemical and cell-based assays to test involvement of RNA "hits" that are identified in our screen. Significance: Association between antisense RNA transcripts and small non-coding RNAs at promoters to control gene expression may represent a novel regulatory mechanism. Understanding its mechanism will extend our understanding of why most of the genome is transcribed but not translated into protein, and how gene expression is regulated by RNA at promoters. This proposal will impact research by demonstrating that RNA can be used to target gene promoters and activate gene expression. The proposed experiments are designed to provide data that will be generally useful for investigators seeking to manipulate other genes in cultured cells and in animal models. PUBLIC HEALTH RELEVANCE: Understanding how non-coding RNAs selectively activate or repress gene expression of gene promoters may reveal a general mechanism of gene regulation that has not been previously appreciated. Many disease states are due to a decrease in the expression of a critical protein. Exploring the capability of RNA to specifically increase expression of a target gene will encourage new approaches in the treatment of disease and in developing therapeutic leads.