Mechanistic Analysis of Intracellular RNA Folding

细胞内RNA折叠的机制分析

基本信息

批准号：
8258778
负责人：
Martha J. Fedor
金额：
$ 38.37万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-03-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8258778
关键词：
Address Affect Area Award Behavior Binding Biogenesis Biological Biological Models Biological Process Catalysis Catalytic RNA Cells Chemicals Communicable Diseases Complex DNA Sequence Rearrangement Disease Equilibrium Exhibits Fluorescence Foundations Functional RNA Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Goals Gram-Positive Bacteria Growth and Development function Hereditary Disease In Vitro Kinetics Libraries Life Ligands Maintenance Measures Mediating Messenger RNA Metabolic Methodology Molecular Biology Molecular Chaperones Molecular Genetics Monitor Outcome Pathway interactions Play Process Proteins RNA RNA Folding RNA-Binding Proteins Reaction Reagent Reporting Ribosomes Role Signal Transduction Structure System Therapeutic Therapeutic Intervention Thermodynamics Translations Work Yeast Model System Yeasts base conformational conversion design in vitro activity in vivo insight metabolomics overexpression programs public health relevance small molecule

项目摘要

DESCRIPTION (provided by applicant): RNAs play key roles in maintenance and expression of genetic information and provide potential targets and reagents for therapeutic intervention in pathological states associated with infectious diseases and hereditary disorders. Assembly of precise RNA structures and RNA-ligand complexes is essential to virtually all RNA-mediated processes. Our goal is to understand how RNAs fold into functional structures in living cells. Direct analysis of RNA folding steps in biological processes is challenging because numerous components interact in complex pathways and many steps intervene between assembly of an RNA structure and execution of its biological function. Catalytic RNAs provide useful model systems for probing folding mechanisms because catalytic activity reports directly and quantitatively on assembly of functional RNA structures. The foundation of our program is a system we developed using RNA catalysis to monitor intracellular assembly of RNA structures that integrates molecular biology and genetics with kinetics and thermodynamics. So far, this unique approach has enabled us to show that some kinetic and equilibrium parameters of intracellular RNA folding reactions agree remarkably well with parameters measured for the same reactions in vitro, provided that in vitro reactions approximate intracellular ionic conditions. On the other hand, we found that competition between alternative RNA secondary structures produces dramatically different outcomes in vitro and in vivo. The discrepancy between in vitro and in vivo RNA folding behavior highlights the importance of investigating RNA folding directly and quantitatively in a biological context. We propose to elaborate upon this approach by incorporating metabolomics and fluorescence methodologies and apply it to study new areas of intracellular RNA folding. These areas include the interplay between kinetics and thermodynamics during mRNA remodeling after transit through the ribosome, the influence of RNA-binding proteins and small ligands on RNA folding during transcription, and the mechanisms through which regulatory RNAs integrate information from multiple chemical signals to regulate gene expression. The proposed studies will generate fundamental insights into folding of the RNA structures that are central to normal growth and development and the assembly of RNA complexes with intracellular ligands that mediate gene regulation. The results of this work will also provide a framework for developing technical and therapeutic applications that involve RNAs as targets and reagents. PUBLIC HEALTH RELEVANCE: RNAs play key roles in maintenance and expression of genetic information and provide potential targets and agents for therapeutic intervention. The proposed studies of intracellular RNA folding and interactions with chemical signals will contribute basic insight into RNA-mediated processes in growth, development, and disease and provide a framework for the rational design of RNA-based therapeutics.

描述（由申请人提供）：RNA在遗传信息的维持和表达中发挥关键作用，并为与传染病和遗传性疾病相关的病理状态的治疗干预提供潜在的靶标和试剂。精确的 RNA 结构和 RNA-配体复合物的组装对于几乎所有 RNA 介导的过程都是至关重要的。我们的目标是了解 RNA 如何折叠成活细胞的功能结构。直接分析生物过程中的 RNA 折叠步骤具有挑战性，因为许多成分在复杂的途径中相互作用，并且在 RNA 结构的组装和其生物功能的执行之间存在许多步骤。催化 RNA 为探测折叠机制提供了有用的模型系统，因为催化活性可以直接定量地报告功能性 RNA 结构的组装。我们项目的基础是我们开发的一个系统，该系统使用 RNA 催化来监测 RNA 结构的细胞内组装，该系统将分子生物学和遗传学与动力学和热力学相结合。到目前为止，这种独特的方法使我们能够证明，细胞内 RNA 折叠反应的一些动力学和平衡参数与体外相同反应测量的参数非常吻合，前提是体外反应接近细胞内离子条件。另一方面，我们发现替代 RNA 二级结构之间的竞争在体外和体内产生显着不同的结果。体外和体内 RNA 折叠行为之间的差异凸显了在生物学背景下直接定量研究 RNA 折叠的重要性。我们建议通过结合代谢组学和荧光方法来详细阐述这种方法，并将其应用于研究细胞内 RNA 折叠的新领域。这些领域包括通过核糖体后 mRNA 重塑过程中动力学和热力学之间的相互作用、转录过程中 RNA 结合蛋白和小配体对 RNA 折叠的影响，以及调节 RNA 整合来自多种化学信号的信息以调节基因的机制。表达。拟议的研究将对正常生长和发育至关重要的RNA结构的折叠以及介导基因调控的细胞内配体的RNA复合物的组装产生基本的见解。这项工作的结果还将提供一个框架，用于开发涉及 RNA 作为靶标和试剂的技术和治疗应用。公共卫生相关性：RNA 在遗传信息的维持和表达中发挥着关键作用，并为治疗干预提供潜在的靶点和药物。拟议的细胞内 RNA 折叠及其与化学信号相互作用的研究将有助于对 RNA 介导的生长、发育和疾病过程的基本了解，并为合理设计基于 RNA 的疗法提供框架。