Poster abstracts
Poster number 38 submitted by Debadrita Paul
A global pulse-labeling approach to investigate flux of nonsense-mediated mRNA decay through alternative routes
Debadrita Paul (Molecular Cellular and Developmental Biology), Zhongxia Yi (Department of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics)
Abstract:
Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic quality control pathway that regulates the expression of both premature termination codon (PTC)-containing transcripts and many normal physiological mRNAs. NMD is activated by core factors UPF1, UPF2, and UPF3, and can be further enhanced when the exon-junction complex (EJC) is present downstream of a PTC. The widely accepted NMD models suggest that UPF3 connects the prematurely terminating ribosome to 3’UTR bound EJC. However, previous studies have shown that UPF3 is dispensable for NMD in human cells, and it functions as an enhancer within a specific branch rather than as an obligatory NMD factor. A key limitation of these studies is their reliance on steady-state mRNA abundance, which cannot distinguish altered mRNA decay from changes in transcription. To address this gap, we are applying a 4-thiouridine (4-sU) pulse-labeling approach to quantify decay rates of PTC-containing versus PTC-free transcripts in WT and UPF3KO HCT116 cells. Through optimization of 4sU concentration, pulse duration, and T-to-C conversion efficiency, we have established conditions to quantify isoform-level decay rates. Using this approach, we will quantify the extent of UPF3-dependent and -independent NMD and identify transcript features that govern UPF3-dependence. Our preliminary work also shows that pharmacological inhibition of the EJC and SMG1, a kinase that phosphorylates core NMD factor UPF1, further attenuates UPF3-dependent NMD. This suggests that key regulators of the pathway can affect NMD independently of UPF3, providing a basis to investigate EJC dependence and UPF1 phosphorylation status across alternative routes of the NMD pathway. Our findings also raise the question of how the EJC is sensed without UPF3, which will be a focus of our future work.
References:
Yi Z, Sanjeev M, Singh G. The Branched Nature of the Nonsense-Mediated mRNA Decay Pathway. Trends Genet. 2021 Feb;37(2):143-159. doi: 10.1016/j.tig.2020.08.010. Epub 2020 Sep 29. PMID: 33008628; PMCID: PMC7854845.
Yi Z, Arvola RM, Myers S, Dilsavor CN, Abu Alhasan R, Carter BN, Patton RD, Bundschuh R, Singh G. Mammalian UPF3A and UPF3B can activate nonsense-mediated mRNA decay independently of their exon junction complex binding. EMBO J. 2022 May 16;41(10):e109202. doi: 10.15252/embj.2021109202. Epub 2022 Apr 22. PMID: 35451102; PMCID: PMC9108626.
Keywords: NMD, 4sU labelling