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

Abstract

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that prevents the accumulation of harmful truncated proteins by degrading transcripts with premature termination codons (PTCs). NMD efficiency varies across many contexts, but the factors that influence this variability remain poorly understood. Here, we find an enrichment of glycine (Gly) codons preceding a PTC in common nonsense variants in contrast with a depletion of Gly codons preceding a normal termination codon (NTC). Gly-PTC contexts have higher NMD activity compared to an alanine-PTC context, and this effect is stronger on NMD substrates with long 3'UTRs. We used a massively parallel reporter assay to test all possible combinations of -2 and -1 codons, the PTC, and the +4 nucleotide to assess comprehensively how PTC sequence context affects NMD efficiency. A random forest classifier revealed that peptidyl-tRNA hydrolysis rate during translation termination was the most important feature in discriminating high and low NMD activity. We show with in vitro biochemical assays that Gly-TC contexts have the slowest termination rate compared to other codons. Furthermore, Gly-PTC enrichment is most pronounced in genes that tolerate loss-of-function variants, suggesting that enhanced NMD of Gly-PTC context has shaped the evolution of PTCs. Based on these findings, we propose that NMD efficiency is modulated by the "window of opportunity" offered by peptidyl tRNA hydrolysis rate and thus, translation termination kinetics.