A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay

Abstract

Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and SMG1i, alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

Systematic analysis of nonsense variants uncovers peptide release rate as a novel modifier of nonsense-mediated mRNA decay

“…EJC-independent NMD is weaker than EJC-enhanced NMD (31,(34)(35)(36) and have different cues. For EJC-independent NMD, a long 3′ UTR downstream of the PTC remains bound by RBPs like UPF1, which would ordinarily be dislodged by the ribosome during translation.…”

“…The cause for this apparent discrepancy in the results from these two analyses is unclear. We know that EJC-independent NMD is weaker than EJC-enhanced NMD (Buhler et al, 2006; Chu et al, 2021; Kolakada et al, 2023; Singh et al, 2008) and has distinct triggers. Thus, the window of opportunity during termination may matter more for a weaker, EJC-independent target, than a stronger EJC-enhanced target.…”

“…To distinguish between these hypotheses, we used a set of NMD reporters (31) that enabled the study of EJC-independent and EJC-enhanced forms of NMD, as well as NMD-associated mechanisms such as PTC readthrough and truncated protein degradation. The "NMD+" reporter consists of a bi-directional promoter that drives BFP as a transfection control, in addition to an context.…”

“…The NMD reporter plasmids have been previously described (31). For individual fluorescent and luminescent reporters containing Gly, Val, Ala, and Tyr amino acids before the PTC, oligos containing these sequences (Integrated DNA Technologies) and matching the EcoRI (NEB, R3101S) and XhoI (NEB, R0146S) restriction sites were synthesized.…”