Structural dynamics of regulatory RNAs and ribonucleoproteins

摘要 RNA是一种结构适应性聚合物，具有复杂的三级结构，并经历了巨大的- 缩放构象变化以调节细胞活性。RNA功能不可或缺的一点是其显著的可塑性 以及响应于包括小分子和蛋白质辅因子的刺激而结构适应的能力。尽管 在生物学中的核心作用，对RNA折叠和分子生物学原理的全面理解， 缺乏认可。这项研究计划解决了这些差距，以了解在原子水平上，RNA如何 在正常细胞功能和疾病状态下折叠并识别结合伴侣。使用RNA适体 和7SK核糖核蛋白（RNP）作为模型体系，我们结合联合收割机溶液核磁共振谱、X射线衍射、 晶体学、质谱学和基础生物化学的多学科方法， 理解RNA结构动力学和分子组装。真核生物7SK RNP是一种主要的 转录调节因子由7SK RNA和蛋白质组分组成，7SK RNP结合并灭活 必需正转录延伸因子B（P-TEF B）的激酶活性。必须释放P-TEFb 从7SK RNP激活RNA聚合酶II转录进行性延伸。P-TEFb失调或 7SK RNP功能障碍与几种遗传疾病有关，包括癌症、心脏病和糖尿病。 原始侏儒症此外，几种病毒操纵宿主7SK RNP用于病毒存活，特别是HIV-1和 最近SARS-CoV-2强调了7SK RNP在生物过程中的重要性。尽管其关键 功能和生物医学意义，目前很少有机制的见解7SK RNP功能在斯塔克 与其他监管RNP相比。这种差距主要是由于缺乏基本的7SK RNP基础知识 它的特点是：7SK RNA如何折叠，蛋白质如何组装到7SK RNA上，以及7SK RNP如何结构化。有 迫切需要回答这些悬而未决的问题，以提供对7SK RNP结构的基本见解 生物学，并且对于全面了解7SK RNP及其在以下方面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簇，阐明7SK RNA构象动力学， 揭示7SK RNP蛋白质组和蛋白质-蛋白质相互作用网络在正常细胞功能， 基因毒性压力下。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方面的知识缺口 organization.这项工作将提供RNA-蛋白质相互作用的基础知识，可以扩展到 理解其他RNP的RNA-蛋白质识别的基本原理。

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.