Establishing Methods to Delineate 3'UTR-mediated Regulation

摘要 基因表达的调节是细胞功能的基础，基因表达的改变是细胞功能的重要组成部分。 人类疾病的常见原因。基因调控通常在转录水平上进行研究，然而， 人们越来越认识到基因表达的转录后调节。内 在mRNA中，决定其转录后命运的大部分序列信息都发生在转录后的细胞内。 3′非翻译区（3 ′ untranslated region，3′UTR）。在人类和其他哺乳动物中，3′ UTR是 通常比其他生物体更大，包含更多保守的序列元件， 预测影响适合度的基因相对于基因组的其他非编码部分在3′ UTR中富集， 包括启动子、增强子、内含子和基因间区。多种调节模式是由 3′ UTR，包括mRNA稳定性和翻译的调节。这项建议的主要目标是制定一项 这是一套系统地确定全长3′UTR序列对所有细胞的影响的测定方法。 转录后基因调控的主要模式。在目标I中，我们将开发能够 平行测量数千个3′ UTR，使用一种新的基于细胞的测定，其中3′UTR报告基因被 整合到基因组中。特别是，我们将确定对转录水平，稳定性， 翻译状态和产生的总蛋白，作为3′UTR序列的功能。除了纯粹的 基因表达的定量控制，越来越清楚的是，一个子集的3′ UTR的功能，以控制 转录本在细胞内的定位。转录本定位的控制可影响转录本稳定性， 翻译，但也可以定位编码的蛋白质。在目标II中，我们将扩展我们的方法来检查 同一组3′UTR的亚细胞定位，侧重于细胞核的差异定位， 细胞质，内质网和RNA颗粒，3′ UTR最有可能 介导亚细胞mRNA定位。总之，这些分析将产生一个全面的， 调控作用的定量定义，这将使我们能够定义3′UTR的作用，几乎在每一个后， 转录过程重要的是，我们开发的工具将特别适合于分析不同的 3′UTR同种型或与疾病有关的人变体。

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.