Establishing Methods to Delineate 3'UTR-mediated Regulation

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ABSTRACT Regulation of gene expression is fundamental to cell function, and alterations in gene expression are a frequent cause of human disease. Gene regulation is typically investigated at the level of transcription, yet there is a growing recognition of consequential post-transcriptional modulation of gene expression. Within mRNAs, much of the sequence information that determines their post-transcriptional fates occurs within a specialized region known as the 3′UTR (3′ untranslated region). In humans and other mammals, 3′UTRs are typically larger and contain more conserved sequence elements than in other organisms, and mutations predicted to impact fitness are enriched in 3′UTRs relative to other noncoding portions of the genome, including promoters, enhancers, introns and intergenic regions. Multiple modes of regulation are elicited by 3′UTRs, including regulation of mRNA stability and translation. The major goal of this proposal is to develop a suite of assays with which to systematically determine the impact of full length 3′UTR sequences upon all major modes of post-transcriptional gene regulation. In Aim I, we will develop high-throughput assays capable of measuring thousands of 3′UTRs in parallel, using a novel cell-based assay in which 3′UTR reporters are integrated into the genome. In particular, we will determine the impact on transcript levels, stability, translational status and overall protein produced, as a function of 3′UTR sequence. In addition to purely quantitative control of gene expression, it is increasingly clear that a subset of 3′UTRs function to control transcript localization within the cell. Control of transcript localization can impact transcript stability and translation, but can also localize the encoded protein. In Aim II, we will extend our methods to examine the sub-cellular localization of the same set of 3′UTR, focusing on differential localization to the nucleus, cytoplasm, endoplasmic reticulum and RNA granules, regions of the cell for which 3′UTRs are most likely to mediate subcellular mRNA localization. Together, these assays will generate a comprehensive and quantitative definition of regulatory effects, which will allow us to define a 3′UTR’s role in almost every post- transcriptional process. Importantly, the tools that we develop will be particularly suitable for assaying different 3′UTR isoforms or human variants implicated in disease.