Structural dynamics of regulatory RNAs and ribonucleoproteins

调节RNA和核糖核蛋白的结构动力学

• 批准号: 10491733
• 负责人: Catherine Eichhorn
• 金额: $ 37.25万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10491733
• 关键词: 2019-nCoV; Address; Adopted; Architecture; Binding; Biochemistry; Biological Models; Biological Process; Biology; Cell physiology; Complex; Disease; Dwarfism; Elements; Foundations; Genetic Diseases; Genetic Transcription; Genotoxic Stress; HIV-1; Health; Heart Diseases; Human; Knowledge; Ligands; Malignant Neoplasms; Mass Spectrum Analysis; Molecular; Molecular Conformation; NMR Spectroscopy; Normal Cell; Phosphotransferases; Polymers; Positive Transcriptional Elongation Factor B; Proteins; Proteome; RNA; RNA Conformation; RNA Folding; RNA Polymerase II; RNA-Binding Proteins; RNA-Protein Interaction; Research; Resolution; Ribonucleoproteins; Role; Specificity; Stimulus; Structure; Viral; Virus; Work; X-Ray Crystallography; aptamer; cofactor; improved; insight; interdisciplinary approach; molecular assembly/self assembly; molecular recognition; programs; protein complex; protein protein interaction; rational design; response; small molecule; structural biology

ABSTRACT RNA is a structurally adaptive polymer that adopts complex tertiary structures and undergoes dramatic large- scale conformational changes to regulate cellular activity. Integral to RNA function is its remarkable plasticity and ability to structurally adapt in response to stimuli including small molecules and protein cofactors. Despite a central role in biology, a comprehensive understanding of the principles that govern RNA folding and molecular recognition is lacking. This research program addresses these gaps to understand, at an atomic level, how RNA folds and recognizes binding partners during normal cell function and in disease states. Using RNA aptamers and the 7SK ribonucleoprotein (RNP) as model systems, we combine solution NMR spectroscopy, X-ray crystallography, mass spectrometry, and basic biochemistry in a multidisciplinary approach to advance understanding of RNA structural dynamics and molecular assembly. The eukaryotic 7SK RNP is a major regulator of transcription. Comprised of the 7SK RNA and protein components, 7SK RNP binds and inactivates the kinase activity of the essential positive transcription elongation factor b (P-TEFb). P-TEFb must be released from the 7SK RNP to activate RNA Polymerase II transcription processive elongation. P-TEFb dysregulation or 7SK RNP malfunction is associated with several genetic diseases including cancers, heart disease, and primordial dwarfism. Moreover, several viruses manipulate host 7SK RNP for viral survival, notably HIV-1 and more recently SARS-CoV-2 underscoring the significance of 7SK RNP in biological processes. Despite its critical function and biomedical significance, there are presently few mechanistic insights into 7SK RNP function in stark contrast to other regulatory RNPs. This gap is largely due to a lack of fundamental knowledge on basic 7SK RNP features: how 7SK RNA folds, how proteins assemble onto 7SK RNA, and how 7SK RNP is structured. There is a critical need to answer these outstanding questions to provide foundational insights into 7SK RNP structural biology, and are essential to achieving a comprehensive understanding of 7SK RNP and its central role in biology. Over the next five years, we will determine high resolution structures of RNA aptamer-ligand complexes, 7SK RNA elements involved in P-TEFb release, and 7SK RNA-protein complexes. We will identify the determinants for RNA-ligand or RNA-protein binding specificity, elucidate 7SK RNA conformational dynamics, and uncover the 7SK RNP proteome and protein-protein interaction network during normal cell function and under genotoxic stress. Long-term, we will use newly gained knowledge to rationally design improved aptamer- ligand pairs, determine global folding and dynamics of 7SK RNA, identify the molecular mechanisms of P-TEFb release from 7SK RNP, and determine the 7SK RNP macromolecular architecture. Findings will address critical knowledge gaps in RNA molecular recognition, 7SK RNA structure, 7SK RNA-protein recognition, and RNP organization. This work will provide fundamental knowledge of RNA-protein interactions that can be extended to understanding the foundational principles of RNA-protein recognition for other RNPs.

抽象的 RNA是一种结构适应性聚合物，采用复杂的三级结构并经历戏剧性的大变化。 规模构象变化来调节细胞活动。 RNA 功能不可或缺的一部分是其卓越的可塑性 以及响应小分子和蛋白质辅助因子等刺激而进行结构适应的能力。尽管有 生物学中的核心作用，全面了解控制 RNA 折叠和分子的原理 缺乏认可。该研究计划解决了这些空白，以在原子水平上了解 RNA 如何 在正常细胞功能和疾病状态下折叠并识别结合伙伴。使用 RNA 适体 和 7SK 核糖核蛋白 (RNP) 作为模型系统，我们结合了溶液核磁共振波谱、X 射线 晶体学、质谱和基础生物化学以多学科方法推进 了解 RNA 结构动力学和分子组装。真核7SK RNP是一个主要的 转录调节因子。 7SK RNP 由 7SK RNA 和蛋白质成分组成，可结合并失活 必需正转录延伸因子 b (P-TEFb) 的激酶活性。 P-TEFb 必须释放 7SK RNP 激活 RNA 聚合酶 II 转录进行性延伸。 P-TEFb 失调或 7SK RNP 功能障碍与多种遗传疾病有关，包括癌症、心脏病和 原始侏儒症。此外，一些病毒操纵宿主 7SK RNP 来维持病毒生存，特别是 HIV-1 和 最近的 SARS-CoV-2 强调了 7SK RNP 在生物过程中的重要性。尽管它很关键 功能和生物医学意义，目前对 7SK RNP 功能的机制了解还很少。 与其他监管 RNP 相比。这种差距很大程度上是由于缺乏对基本 7SK RNP 的基础知识造成的 特征：7SK RNA 如何折叠、蛋白质如何组装到 7SK RNA 上以及 7SK RNP 如何构建。有 迫切需要回答这些悬而未决的问题，以便为 7SK RNP 结构提供基础见解 生物学，对于全面了解 7SK RNP 及其在生物学中的核心作用至关重要 生物学。在接下来的五年里，我们将确定RNA适体-配体复合物的高分辨率结构， 参与 P-TEFb 释放的 7SK RNA 元件和 7SK RNA-蛋白质复合物。我们将确定 RNA-配体或 RNA-蛋白质结合特异性的决定因素，阐明 7SK RNA 构象动力学， 揭示正常细胞功能期间的 7SK RNP 蛋白质组和蛋白质-蛋白质相互作用网络 在遗传毒性应激下。从长远来看，我们将利用新获得的知识来合理设计改进的核酸适体- 配体对，确定 7SK RNA 的整体折叠和动力学，确定 P-TEFb 的分子机制 从7SK RNP中释放，并确定7SK RNP大分子结构。调查结果将解决关键问题 RNA 分子识别、7SK RNA 结构、7SK RNA 蛋白质识别和 RNP 方面的知识差距 组织。这项工作将提供 RNA-蛋白质相互作用的基础知识，这些知识可以扩展到 了解其他 RNP 的 RNA 蛋白质识别的基本原理。