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Peptidyl-tRNA hydrolysis rate influences the efficiency 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 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE261151 | GEO | 2025/03/10

REPOSITORIES: GEO

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Project description:We used a modification of GINI analysis to identify genes containing premature translation termination codons (PTC) generated by nonsense or frameshift mutations in prostate cancer cell lines. The analysis was performed in two steps. In the first step nonsense mediated mRNA decay (NMD) was inhibited in prostate cancer cell lines using incubation with medium containing caffeine for 4 hours. Gene expression analysis of caffeine treated or untreated cells after this step detects mRNA accumulation that takes place for genes containing PTC and as well as for genes that show induction of transciption due to stress caused by NMD inhibition. In the second step either both transcription and NMD or transcription only are blocked by incubating cell in a medium containing either both actinomycin D and caffeine or actinomacin D only for 4 hours. Gene expression analysis after this second step detects mRNA degradation for genes containing PTC as well as for genes that show induction of transciption due to stress caused by NMD inhibition. The efficiency of mRNA degradation for genes containing PTC during this step depends on whether NMD is inhibited or not. The efficiency of mRNA degradation for stress response genes does not depend on whether NMD is inhibited or not. Our modified GINI protocol allows efficient separation of genes containing PTC from the genes that show mRNA increases due to stress response to NMD inhibition. Experiment Overall Design: The LNCaP and 22Rv1 cells were seeded into four 60 mm tissue culture plates each. Caffeine (10 mM) was added to three plates and one plate was incubated without caffeine as a control. Following four hours? incubation, the medium was removed from one plate (with caffeine) and from the control plate and total RNA was prepared using TRIZOL reagent, according to manufacturer?s instructions, and used for an Affymetrix U133Plus2.0 oligonucleotide array hybridization. The caffeine-containing medium of the two remaining plates was removed, the cells were washed twice with phosphate-buffered saline and the medium with actinomycin D (2 mkg/ml) together with caffeine (10 mM) was added to one plate. Actinomycin D alone was added to the other plate. Following another four hours? incubation, the total RNA from both plates was prepared and used for an Affymetrix U133Plus2.0 oligonucleotide array analysis.

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Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay

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