Poster abstracts
Poster number 49 submitted by Zhongxia Yi
Investigating the mechanism of UPF3B-independent nonsense-mediated mRNA decay
Zhongxia Yi (Department of Molecular Genetics), Lauren A. Woodward (Department of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics)
Abstract:
Nonsense-mediated mRNA decay (NMD) degrades aberrant transcripts with premature termination codons to limit the production of truncated polypeptides. NMD also regulates 10-20% of normal transcripts in mammals, and hence, it is essential for processes such as development and stress response. All three core NMD factors, Upf1p, Upf2p and Upf3p, are required for NMD in yeast, whereas UPF3B is dispensable for NMD of several mRNAs in human cells. The mechanism of such UPF3B-independent NMD and mRNAs regulated by this distinct NMD branch remain largely unknown. In contrast to yeast, recognition of NMD substrates in human cells is assisted by the exon junction complex (EJC), which is deposited at exon-exon junctions by the spliceosome. The EJC facilitates mRNA export to the cytoplasm, where it is removed from mRNAs by the first translating ribosome. If an EJC remains bound downstream of a terminated ribosome, it signals premature termination by recruiting NMD factors UPF3B and UPF2 to activate UPF1 and trigger rapid mRNA degradation. To investigate UPF3B-independent NMD mechanism, we have used CRISPR-Cas9 to create UPF3B knockout in a colorectal carcinoma HCT116 cell line. RNA-seq from the knockout and control cells have identified global mRNA targets of UPF3B-dependent and -independent NMD. Our data show that in HCT116 cells most NMD targets can undergo UPF3B-independent NMD and UPF3B only regulates a small subset of mRNAs. To further discriminate between UPF3B-dependent and -independent mRNA targets, we have inserted a FLAG affinity tag into the UPF3B gene to identify UPF3B-bound mRNA targets via RIP-seq. We have also discovered that UPF2, but not UPF3B, associates with the EJC-containing peripheral EJC factor RNPS1, suggesting that this EJC factor may operate within the UPF3B-independent NMD branch. Consistently, RNPS1 knockdown leads to upregulation of several UPF3B-independent NMD target mRNAs. We have inserted FLAG tag into UPF1 in both wildtype and UPF3B knockout cell lines. FLAG-UPF1 immunoprecipitation from these cells will reveal UPF3B-dependent and -independent NMD complexes. Overall, our work will elucidate the mechanisms of how parallel NMD branches regulate different mRNA subsets and potentially distinct biological processes.
Keywords: Nonsense-mediated mRNA decay, UPF3B, Exon junction complex