RNA functions as the central conduit of information exchange in all cells, a role encapsulated in two critical observations. First, a large fraction of emerging infectious diseases are caused by RNA viruses including Ebola, Chikungunya, Zika, and Dengue. Second, a much larger fraction of the mammalian genome is transcribed into diverse kinds of non-coding RNAs (~70%) than is translated into protein (1- 2%). The functions of messenger, non-coding, and viral RNAs are governed by the linear sequence, base-paired secondary structure, higher-order tertiary structure, and quaternary interactions involving proteins and small molecules. Overall, our understanding of the number and complexity of RNA structures and how RNA structure drives diverse biological functions is very limited. Most methods developed to date for analyzing RNA structure in high-throughput ways do not measure structure in a definitive and accurate way, making it difficult to define broad principles for interrelationships between RNA structure and function. We seek to understand the fundamental roles of RNA structure in all areas of biology by pursuing a two-pronged approach involving (1) inventing, developing, and rigorously validating highly accurate chemistry-based technologies for discovery of novel RNA structures and the networks of interactions between RNAs and proteins and then (2) applying these technologies to problems of broad importance. Here we propose to interrogate the structures and interaction partners of the pathogenic Dengue RNA virus and the Xist long non-coding RNA. Throughout this work, we will focus on in-cell analysis of native viral and endogenous RNAs. This work is expected to have long-term impact for three broad reasons. First, RNA elements with higher-order folds and extensive protein networks are likely to be harbingers of function. Second, there are likely to be structural folds that are different from the relatively limited classes of structures that have been analyzed to date. Third, RNA elements with higher-order folds also contain clefts and crevices that are ideal targets for small- molecule ligands ? and novel drugs ? that modulate biological function by targeting RNA.
This proposal focuses on the broad visions of (i) creating novel technologies for rigorous, quantitative, and experimentally concise analysis of higher-order RNA structures and (ii) applying these technologies to the compelling challenges of discovering novel tertiary structures and protein regulatory networks in the genomes of pathogenic viruses and in non-coding RNAs. This project will create new technologies for analysis of RNA structure and function, ultimately driving both basic biological discovery and novel, RNA-targeted, small-molecule drug development.
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