Establishing Methods to Delineate 3'UTR-mediated Regulation

建立描述 3UTR 介导调节的方法

• 批准号: 10316261
• 负责人: ANDREW W GRIMSON
• 金额: $ 27.78万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-09 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10316261
• 关键词: 3' Untranslated Regions; Biological; Biological Assay; Cell Nucleus; Cell physiology; Cells; Cellular Assay; Code; Collection; Consequentialism; Conserved Sequence; Cytoplasm; Cytoplasmic Granules; Discrimination; Disease; Elements; Endoplasmic Reticulum; Enhancers; Gene Abnormality; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genetic Translation; Genome; Genomics; Goals; Human; Human Biology; Human Cell Line; Human Genome; Intercistronic Region; Introns; Length; Libraries; Location; Mammals; Measurement; Measures; Mediating; Messenger RNA; Methods; Modernization; Mutation; Nucleic Acid Regulatory Sequences; Organism; Output; Post-Transcriptional Regulation; Process; Protein Isoforms; Proteins; RNA; Regulation; Regulatory Element; Reporter; Research; Role; Site; Source; Specific qualifier value; System; Technology; Transcript; Transcription Process; Translations; Untranslated RNA; Untranslated Regions; Variant; base; biological systems; design; fitness; genome-wide; high throughput screening; human disease; improved; mRNA Decay; mRNA Stability; mammalian genome; neglect; next generation sequencing; novel; novel strategies; promoter; tool; virtual

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.

抽象的 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基础。 人类疾病的常见原因通常是在转录水平上进行研究。 人们越来越认识到基因表达的转录后调节。 mRNA，决定其转录后命运的许多序列信息发生在 在人类和其他哺乳动物中，3'UTR 是称为 3'UTR（3' 非翻译区）的特殊区域。 通常比其他生物体更大并且包含更多保守的序列元件和突变 相对于基因组的其他非编码部分，预计会影响适应性的基因在 3'UTR 中富集， 包括启动子、增强子、内含子和基因间区域。 3'UTR，包括 mRNA 稳定性和翻译的调节 该提案的主要目标是开发一种 一套测定法，用于系统地确定全长 3'UTR 序列对所有 在目标 I 中，我们将开发能够进行高通量检测的方法。 使用一种新颖的基于细胞的检测方法并行测量数千个 3'UTR，其中 3'UTR 生产者 特别是，我们将确定对转录水平、稳定性的影响。 除了纯粹的 3'UTR 序列之外，翻译状态和产生的总体蛋白质也是如此。 基因表达的定量控制，越来越清楚的是，3'UTR 的一个子集具有控制基因表达的功能 细胞内转录本定位的控制可以影响转录本稳定性和 翻译，但也可以定位编码的蛋白质，在 Aim 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，重点关注细胞核的差异定位， 细胞质、内质网和 RNA 颗粒，3'UTR 最有可能作用的细胞区域 这些测定共同介导亚细胞 mRNA 定位。 监管效应的定量定义，这将使我们能够定义 3'UTR 在几乎所有后监管中的作用 重要的是，我们开发的工具特别适合分析不同的转录过程。 与疾病有关的 3'UTR 同工型或人类变体。