Poster abstracts
Poster number 102 submitted by Debadrita Paul
Measurement of mRNA decay kinetics to investigate contributions of factor specificity and transcriptional regulation to NMD
Debadrita Paul (Molecular Cellular and Developmental Biology, The Ohio State University), Ren Arvola (Department of Molecular Genetics, The Ohio State University), Zhongxia Yi (Department of Molecular Genetics, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, The Ohio State University)
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. UPF2 and UPF3, although essential in yeast appear dispensable in vertebrates, suggesting they act primarily as enhancers within a branched pathway. However, the relative contribution of different activation routes and factor requirements in human cells remains unclear. Previous studies have relied on knockdown or knockout of NMD factors followed by measurement of steady-state RNA levels to infer NMD efficiency, but these methods have several limitations. Protein knockdowns are incomplete and leave basal protein levels that can sustain NMD, while permanent knockouts can cause lethality or compensatory changes in cells. Moreover, RNA abundance measurement alone cannot disentangle altered mRNA decay from reduced transcription. To address these challenges, we are applying 4-thiouridine pulse-labeling based approaches to quantify isoform-specific decay rates of PTC-containing versus PTC-free mRNAs. We have also generated HCT116 cells expressing auxin-inducible degron-tagged UPF2 and UPF3B, enabling rapid and reversible protein depletion. These tools, along with the available UPF3 knockout HCT116 cell lines, will enable us to investigate NMD kinetics upon acute versus long-term depletion of UPF2 and UPF3. We are also applying the metabolic labeling-based decay assay to test an unexpected link our lab has uncovered between UPF3 and the Super Elongation Complex (SEC), a regulator of promoter-proximal pausing of RNA Pol II. By integrating decay measurements upon perturbation of SEC, we aim to directly assess how transcriptional control feeds into NMD pathway. Together, these studies will establish a unified platform to define both factor-specific and transcriptional determinants of NMD in human cells.
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: nonsense mediated decay, 4sU-based decay kinetics, UPF3