Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay
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 activity varies across many contexts, with the variation having critical consequences during both normal development and disease. However, the factors that influence this variability remain poorly understood. Here, we investigate the relationship between the immediate sequence context of a PTC and NMD activity. 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). Furthermore, Gly-PTC enrichment is most pronounced in genes that tolerate loss-of-function variants, suggesting a biological impact for Gly-PTC contexts. Consistent with this idea, Gly-PTC contexts have higher NMD activity compared to an alanine-PTC context. We used a massively parallel reporter assay to comprehensively assess the effect of PTC sequence context on NMD activity, which revealed that peptidyl-tRNA hydrolysis rate during translation termination was the most important feature in discriminating high and low NMD activity. We show with biochemical assays in a eukaryotic system that Gly-TC contexts have the slowest termination rate compared to other codons. Based on these findings, we propose that NMD activity is modulated by the "window of opportunity" offered by peptidyl-tRNA hydrolysis rate and thus, translation termination kinetics. Gly-PTC contexts may have coevolved with NMD to ensure robust elimination of potentially toxic truncated proteins from non-essential genes. Overall design: To investigate the effect of PTC sequence context on EJC-independent and EJC-enhanced NMD targets, we integrated all possible combinations of the PTC Sequence context (NNNNNNTRRN, where TRR is either one of the stop codons or a TGG no NMD control) of previously validated EJC-independent and EJC-enhanced NMD reporters We integrated these reporter libraries into HEK293T cells via Cre-Lox recombination. We treated the cells with DMSO or SMG1i (an NMD inhibitor) for 24 hours and harvested total RNA. The RNA was converted into cDNA, after which libraries were made and sent for sequencing.