Structural dynamics of regulatory RNAs and ribonucleoproteins

摘要

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.