Date of this Version
Mohamed, Amr. NMR Structural Studies of RBM7 Protein and Insights into 7SK RNA-RBM7 Interactions, Involved in DNA Damage Response. Undergraduate Honors Thesis. University of Nebraska-Lincoln. 2022.
Living organisms are continuously undergoing DNA damage through exposure to chemical and environmental agents, and cells have adapted sophisticated pathways to regulate transcription to respond to DNA damage. One of these pathways is the 7SK RNP, which was recently reported to bind to the protein RNA Binding Motif 7 (RBM7) to turn on the transcription of genes involved in cell survival upon DNA damage. To achieve this regulatory function, RBM7 interacts with 7SK RNA via its RNA Recognition Motif (RRM) as well as core 7SK RNP proteins. However, little is known about how RBM7 interacts with the 7SK RNP upon DNA damage, in particular the mode of RNA recognition by the RRM. To study the 7SK RNA-RBM7 interactions, RBM7 constructs with different C-terminal boundaries were generated and characterized using solution Nuclear Magnetic Resonance (NMR) spectroscopy. Individual-nucleotide resolution UV Crosslinking and ImmunoPrecipitation (iCLIP) sequencing data collected and deposited in a previous study was re-analyzed to identify nucleotide-resolution crosslinking to stem loop 3 (SL3) of 7SK RNA. Finally, binding assays and solution NMR experiments of RBM7 RRM constructs and 7SK SL3 were performed to determine the RBM7 binding to 7SK SL3 in vitro. We found that RBM7 binds weakly to 7SK SL3 RNA and specific residues in the RNP1 and RNP2 of RBM7 binds to 7SK SL3 construct. Together, these results provide a foundation for in vitro and in vivo studies to understand RBM7-7SK RNP interactions and RBM7 regulatory function upon DNA damage.