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A 经常引起人类疾病的原因。通常在转录水平上研究基因调控 对基因表达的转录后调节的认识越来越多。之内 mRNA，确定其转录后命运的许多序列信息发生在 专业区域称为3'UTR（3'未翻译区域）。在人类和其他哺乳动物中，3'utrs是 通常比其他生物更大，并且包含更多的组成序列元素和突变 相对于基因组的其他非编码部分，预测的影响适应性富含3'UTRS， 包括启动子，增强子，内含子和基因间区域。多种监管模式由 3'Utrs，包括调节mRNA稳定性和翻译。该提议的主要目标是开发 可以系统地确定全长3'UTR序列对所有的测定法的套件 转录后基因调控的主要模式。在AIM I中，我们将开发能够的高通量测定法 使用3'UTR记者的新型基于细胞的测定法，并并行测量数千个3'Utrs 集成到基因组中。特别是，我们将确定对转录水平，稳定性的影响 产生的翻译状态和总体蛋白质是3'UTR序列的函数。除了纯粹 基因表达的定量控制，越来越明显的是，3'UTRS功能的子集可控制 在单元格中的转录本定位。转录本定位的控制可能会影响转录本的稳定性和 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以检查 同一组3'UTR的亚细胞定位，重点是对核的差分定位， 细胞质，内质网和RNA颗粒，该细胞的区域最有可能3'UTRS 介导亚细胞mRNA定位。这些测定将共同产生全面的 监管效应的定量定义，这将使我们能够在几乎每个后的每个后 转录过程。重要的是，我们开发的工具将特别适合分析不同的工具 3'UTR同工型或疾病中实施的人类变体。