Investigating novel roles of UPF3 interactions in Nonsense Mediated mRNA Decay
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Date
2024-05
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The Ohio State University
Abstract
RNA quality control is important in maintaining cellular processes and preventing disease. Nonsense mediated mRNA decay (NMD) is a conserved pathway in eukaryotes that proofreads messenger RNA (mRNA) by recognizing premature termination codons (PTCs), preventing the accumulation of deleterious effects of toxic peptides. The UPF3 protein is a core factor in NMD with two paralogs in vertebrates: UPF3A and UPF3B. The paralogs play partially redundant roles in the NMD pathway, with UPF3A being a weaker activator. Previous work in our lab has shown that the middle or “mid” domain of UPF3 underlies differential NMD activity between the paralogs. The UPF3 paralogs also diverge in their physiological roles. UPF3B knockout mice exhibit defects in neuronal development whereas knockout of UPF3A knockout is embryonic lethal. Furthermore, mutations in human UPF3B are associated with intellectual disability, autism, and schizophrenia. Despite UPF3 being an integral NMD factor, its interactions and how these differ between paralogs are not fully understood. Using proteomics and cell based immunoprecipitation in HCT116 cells, our lab has identified novel binding partners of UPF3A and UPF3B. Using FLAG Immunoprecipitation (FLAG-IP), we verified interactions with transcriptional regulator RBBP5, and nucleocytoplasmic shuttling factors, ALKBH5 and KPNA3. Using FLAG-IP and Western blotting, I found that the mid domain did not appear to underlie differential binding of ALKBH5 between UPFA and UPF3B. Finally, we used siRNA mediated knockdown in HCT116 WT cells and RT-qPCR of known NMD targets to deplete UPF3 binding partners and measure their effect on NMD of endogenous PTC-containing mRNAs. Interestingly, in preliminary experiments depletion of KPNA3 and transcriptional elongation regulator, MLLT1 led to reduced levels of PTC-containing isoforms of endogenous NMD targets, but not their PTC-less counterparts. Future work will continue to determine whether UPF3 binding partners affect NMD efficiency, and whether these interactions are critical for UPF3 function in the NMD pathway.
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Keywords
UPF3, RNA quality control, Nonsense Mediated mRNA Decay, UPF3 paralogs