Molecular Switches: Regulatory RNAs Targeting Nuclear Hormone Receptor Promoters

描述(申请人提供)：转录组分析的最新结果表明，基因组的大部分是转录的，RNA转录本是由两条DNA链产生的。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，非编码的RNA转录本与小的非编码的反基因RNA和蛋白质在基因启动子上相互作用来调节表达。启动子上的非编码RNA之间的联合控制基因表达可能代表了一种新的调控机制。目的：我们的主要目标是通过鉴定相关蛋白，并确定它们如何与启动子DNA和RNA转录本相互作用，进一步阐明抗基因RNAs激活或抑制PR基因表达的机制。将进行基于细胞的分析以确定涉及的蛋白质。为了了解蛋白质-核酸的相互作用，我们将使用旨在分离PR基因内与启动子DNA结合的特定蛋白质或PR基因产生的RNA转录本的方法。这些数据将使我们能够建立一个机制模型，用于RNA介导的基因表达控制在基因启动子。我们的第二个目标是测试激活agRNAs是否通过诱导基因转录、增强过程延伸和/或稳定RNA转录来增加基因表达。测试agRNA对这些机制的介导作用将有助于理解agRNA作用机制的关键方面。我们的第三个第二个目标(目标III)是研究调节PR基因的内源性抗基因RNA的存在。为了识别潜在的抗基因RNA，我们将使用生物信息学方法来筛选与PR基因启动子内的位置互补的序列的microRNA文库。我们将使用生化和基于细胞的分析来测试在我们的屏幕上识别的RNA“点击”的参与程度。意义：反义RNA转录本和启动子上的小非编码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。了解其机制将扩大我们对为什么大多数基因组被转录而不是翻译成蛋白质，以及基因表达是如何在启动子上受RNA调控的理解。这一提议将影响研究，因为它证明了RNA可以被用于靶向基因启动子和激活基因表达。拟议中的实验旨在为寻求在培养细胞和动物模型中操纵其他基因的研究人员提供普遍有用的数据。

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.