Investigating PYM1 function in cellular and flaviviral gene expression
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Date
2023-05
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The Ohio State University
Abstract
Post-transcriptional regulation of RNAs in the cell is critical to proper expression of the genome. The Exon Junction Complex (EJC) is an RNA binding protein complex that has important role in post-transcriptional regulation of mRNAs. An EJC is deposited on mRNA by the spliceosome ~24 nucleotides upstream of exon-exon junctions (canonical position) to serve as an architectural marker of intron positions. Presence of an EJC on mRNA can enhance its expression by coupling splicing to mRNA processing activities. Further, presence of an EJC after a stop codon can mark an mRNA for degradation through a process called nonsense mediated decay (NMD). NMD is an essential pathway in eukaryotic cells that degrades premature termination codon (PTC)-containing mRNAs and also regulates expression of many normal mRNAs. Our unpublished work suggests that PYM1, a protein that can interact with the ribosome and EJC, may act as an EJC destabilizing factor, preventing EJC accumulation at positions away from exon-exon junctions (non-canonical position). Loss of EJC-PYM1 interaction does not alter ribosome-dependent EJC disassembly but increases EJC detection away from exon junctions at non-canonical sites on spliced mRNAs and lncRNAs. Upon loss of PYM1-EJC interaction, non-canonical EJC binding is also greatly increased on intronless RNAs, which do not normally have bound EJCs. This has potential implications for the understanding of gene expression of flaviviruses (e.g., Zika virus, West Nile virus), as PYM1 is reported to act as an anti-viral factor that is hijacked through interaction with flavivirial capsid proteins. This interaction may sequester PYM1 away from its role as an EJC destabilizing factor and allow EJC accumulation on the intronless flaviviral RNAs. This could influence flaviviral translation and/or NMD. In this work, I investigated the molecular basis of PYM1-flaviral capsid protein interaction, the effects of PYM1 depletion on nuclear functions of the EJC and NMD, as well as the potential translational effects of non-canonical EJC deposition on intronless RNAs. Although I was unable to reproduce the flavivirus capsid protein interaction, I was able to determine that PYM1 depletion affects NMD, but not splicing. I also showed that there is no significance difference between intron-less luciferase mRNA expression in control versus PYM1 knock down cells. These findings support our model of PYM1 as a destabilizing factor of EJCs.
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Keywords
mRNA, NMD, EJC, PYM, Flavivirus