Mechanistic Analysis of Intracellular RNA Folding

描述(由申请人提供)：RNA在维持和表达遗传信息方面发挥关键作用，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点和试剂。精确的RNA结构和RNA配体复合体的组装对于几乎所有的RNA介导的过程都是必不可少的。我们的目标是了解RNA如何折叠成活细胞中的功能结构。直接分析生物过程中的RNA折叠步骤是具有挑战性的，因为许多成分以复杂的途径相互作用，并且在RNA结构的组装和其生物学功能的执行之间存在许多步骤。催化RNA为探索折叠机制提供了有用的模型系统，因为催化活性直接和定量地报告了功能RNA结构的组装。我们计划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结构在细胞内的组装，该系统将分子生物学和遗传学与动力学和热力学结合在一起。到目前为止，这一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，不同的RNA二级结构之间的竞争在体外和体内产生了截然不同的结果。体外和体内RNA折叠行为之间的差异突显了在生物学背景下直接和定量研究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阐述这一方法，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，RNA结合蛋白和小配体对转录过程中RNA折叠的影响，以及调控RNA整合来自多个化学信号的信息以调节基因表达的机制。拟议的研究将产生对正常生长和发育至关重要的RNA结构的折叠以及RNA复合体与介导基因调控的细胞内配体的组装的基本见解。这项工作的结果还将为开发以RNA为靶标和试剂的技术和治疗应用提供一个框架。

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.