Formation and regulation of the translating mRNP

翻译 mRNP 的形成和调节

• 批准号: 9136237
• 负责人: David Eric Weinberg
• 金额: $ 39.63万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-19 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9136237
• 关键词: Adopted; Affect; Affinity; Affinity Chromatography; Binding; Binding Proteins; Biochemical; Biological Assay; Cells; Chemicals; Client; Collection; Complex; Data; Disease; Environment; Eukaryota; Gene Expression Regulation; Genes; Goals; Growth and Development function; Health; High-Throughput Nucleotide Sequencing; In Vitro; Individual; Knowledge; Light; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Messenger RNA; Methods; Modeling; Molecular; Molecular Analysis; Molecular Conformation; Monitor; Mus; Mutation; Organism; Output; Peptide Initiation Factors; Poly(A) Tail; Poly(A)-Binding Proteins; Positioning Attribute; Process; Protein Biosynthesis; Proteins; Protocols documentation; RNA-Protein Interaction; Reagent; Regulation; Reporter; Research; Resolution; Ribosomes; Role; Scanning; Structure; System; Tail; Techniques; Testing; Translating; Translation Initiation; Translation Process; Translations; Work; Yeasts; base; cell growth; cell type; crosslink; crosslinking and immunoprecipitation sequencing; embryonic stem cell; helicase; human disease; improved; in vivo; inhibitor/antagonist; insight; mRNA Stability; messenger ribonucleoprotein; mutant; novel; novel strategies; particle; protein protein interaction; research study; reverse genetics; tool; transcriptome; translation factor

DESCRIPTION (provided by applicant): Translational control is a major mode of gene regulation in eukaryotes that is often perturbed in human diseases. Despite extensive mechanistic studies of translation, it has not been possible to monitor in vivo most steps of translation initiation, which is the primary point of translational control. As a result, how endogenous translating messenger ribonucleoprotein particles (mRNPs) are formed and regulated in the competitive cellular environment is not completely understood. The goal of this application is to develop and apply in vivo methods to study the complex process of translation initiation. By revealing fundamental mechanisms of translational control, the proposed studies will provide new insights into how aberrant translation contributes to diseases, particularly cancer. In addition, the experimental systems established in this application can be widely adapted to studying translation initiation in different cellular and disease states. The methods developed in this application are based on the premise that the translating mRNP contains many mRNA-binding proteins, so individual steps of initiation can be studied by identifying and characterizing the underlying mRNA-protein interactions. This novel approach to the translating mRNP leverages in vivo crosslinking to capture RNA- protein interactions as they occur in the cell, and high-throughput sequencing to identify these interactions across the transcriptome. The experiments proposed in this application will investigate three aspects of the translating mRNP in yeast and mammalian cells. The first specific aim focuses on how the closed-loop structure is formed. Translating mRNPs adopt a circular conformation that enhances translation and mRNA stability, but how this closed-loop structure initially forms is not understood. High-throughput methods will be developed to detect endogenous closed-loop structures in yeast and mouse embryonic stem cells (mESCs). These methods will be combined with reverse genetics, chemical inhibitors, and reporter assays to determine how known protein-protein interactions and the process of translation itself contribute to closed-loop formation. These studies will provide a technical and conceptual framework for molecular analysis of the closed loop in vivo. The second major aim is to identify what determines the efficiency with which mRNAs are translated, which varies widely among endogenous genes for largely unknown reasons. The studies in this application will test the hypothesis that translational efficiency (TE) is largely explained by recruitment of the eIF4F cap- binding complex, which is the first step of initiation. Transcriptome-wide measurements of eIF4F binding and TE, combined with biochemical analysis of eIF4F-mRNA interactions, will be used to evaluate this hypothesis. Comparing results obtained in yeast, mESCs, and embryoid bodies (derived from mESCs) will reveal the extent to which principles of translational control differ between organisms and between cell types. The third aim of this project is to study the scanning 40S ribosome, which has not previously been observed or directly assayed. A method will be developed to identify the positions of isolated 40S ribosomes across the mESC transcriptome. Examining how scanning ribosomes are affected by chemical inhibition of the eIF4A helicase will provide new insights into the molecular details of scanning and the role of eIF4A in this process. Collectively, these aims will improve our knowledge of cellular translation mechanisms and our understanding of how these mechanisms go awry in disease.

描述（由申请人提供）：翻译控制是真核生物中基因调节的一种主要模式，通常在人类疾病中受到干扰。尽管经过广泛的翻译机械研究，但不可能在体内监测翻译起始的大多数步骤，这是翻译控制的主要点。结果，在竞争性的细胞环境中形成和调节了内源性翻译信使核糖核蛋白颗粒（MRNP）。该应用的目的是开发和应用体内方法来研究复杂的翻译启动过程。通过揭示转化控制的基本机制，拟议的研究将提供新的见解，以了解异常翻译如何促进疾病，尤其是癌症。此外，在本应用中建立的实验系统可以广泛适应不同的细胞和疾病状态的翻译起始。本应用程序中开发的方法基于以下前提：翻译MRNP包含许多mRNA结合蛋白，因此可以通过识别和表征基本的mRNA-蛋白质相互作用来研究单个启动步骤。这种新颖的方法是在体内交联中翻译MRNP在细胞中发生的RNA蛋白相互作用以及高通量测序以识别整个转录组中的这些相互作用。本应用程序中提出的实验将研究酵母和哺乳动物细胞中翻译MRNP的三个方面。第一个特定目的侧重于如何形成闭环结构。翻译mRNP的采用圆形构象增强了翻译和mRNA稳定性，但是该闭环结构最初是如何理解的。将开发高通量方法来检测酵母和小鼠胚胎干细胞（MESC）中的内源性闭环结构。这些方法将与反向遗传学，化学抑制剂和记者测定法相结合，以确定已知的蛋白质蛋白相互作用以及翻译本身如何有助于闭环形成。这些研究将提供一个技术和概念框架，用于对体内封闭环的分子分析。第二个主要目的是确定什么决定了mRNA翻译的效率，由于未知原因，在内源基因之间有很大变化。本应用中的研究将检验以下假设：转化效率（TE）在很大程度上通过募集EIF4F封闭结合复合物来解释，这是启动的第一步。 EIF4F结合和TE的全转录组测量结果与EIF4F-MRNA相互作用的生化分析相结合，将用于评估该假设。比较在酵母，MESC和胚胎体（源自MESC）中获得的结果将揭示转化控制原理在生物体之间以及细胞类型之间的不同程度。该项目的第三个目的是研究扫描40S核糖体，以前尚未观察到或直接测定。将开发一种方法来识别MESC转录组跨越40S核糖体的位置。检查扫描核糖体如何受到EIF4A解旋酶的化学抑制作用，将为扫描的分子细节和EIF4A在此过程中的作用提供新的见解。总的来说，这些目标将提高我们对细胞翻译机制的了解，以及我们对这些机制如何在疾病中出现问题的理解。