CAREER: Molecular mechanisms of ribonucleoprotein assembly

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Ribonucleic acid (RNA) is an essential biological molecule that performs diverse roles in cellular processes. To carry out its function, RNA must first fold into the correct structure and assemble with required cofactors such as proteins. Despite having a central role in biology, a comprehensive understanding of how RNA folds and binds to protein is lacking. To address these gaps in knowledge, this project uses the 7SK ribonucleoprotein (RNP) complex, a crucial regulator of cell function, as a model system. In addition, this project will characterize folding of synthetic RNAs that bind dyes to become fluorescent that, when tagged onto biologically important RNAs, can be used to observe RNA in live cells. Results arising from the project will provide fundamental insights into RNA structure-function relationships for RNAs involved in biological processes and produce general rules for RNP assembly. The project entwines education alongside research to increase retention and diversity of the next generation of scientists. This project will develop educational activities including incorporating discovery-based chemical biology laboratory coursework into the undergraduate curriculum. Hands-on workshops will be held in partnership with established programs at the University of Nebraska – Lincoln to engage the public in RNA science. RNPs are essential for cell function, yet without comprehensive knowledge of RNA structure and RNP assembly the molecular mechanisms directing cell function cannot be fully understood. The 7SK RNP, a major regulator of eukaryotic transcription, is an ideal model system to advance limited knowledge of RNA-regulated cell function. A fundamental understanding of 7SK RNP structure and assembly will advance understanding of transcription regulation and assembly of other regulatory RNPs; however, there are significant gaps in understanding how 7SK RNA folds and assembles into a functional RNP. To overcome these gaps, this project employs multidisciplinary approaches to determine 7SK RNA structural dynamics, characterize 7SK RNA-protein interactions important for regulatory function; and integrate a fluorescent RNA aptamer into 7SK RNA for live cell imaging in vivo. To achieve these objectives, combined solution NMR spectroscopy, X-ray crystallography, cellular approaches, binding assays, and fluorescence microscopy methods will be used. This project will elucidate the molecular mechanisms of RNA folding and RNP assembly using integrated biophysical and chemical biology approaches, in concert with education activities to advance and support underrepresented communities in STEM. This project is jointly supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences and the Established Program to Stimulate Competitive Research (EPSCoR).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.