Molecular Switches: Regulatory RNAs Targeting Nuclear Hormone Receptor Promoters

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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.