Mistranslation from endogenous tRNA variants in human pan-genome cell lines

Errors in protein synthesis are thought to be rare, yet cells representing the diversity of life tolerate greatly elevated levels of error. One source of translation error in humans are anticodon mutations in transfer RNAs (tRNAs). We found a tRNA Ser AGA -2-3 variant (G35A) that occurs in 2% of the human population causes mis-incorporation of serine (Ser) at phenylalanine (Phe) codons. We developed a dual fluorescent reporter to quantify mis-incorporation levels in live cells, including β-lymphocyte lines from the 1000 genomes project with wild-type or mutant A35 alleles. The mutant cells showed reduced viability, and tRNA sequencing confirmed expression, hypo-modification of C32, and 5’- fragment generation of the mutant tRNA Ser AAA . Transfection with our fluorescent reporter and mass spectrometry confirmed Ser mis-incorporation. The data demonstrate that a natural human genome encoded tRNA mutant causes mistranslation. Our findings have important implications for translation fidelity in humans and the application of missense suppressor tRNAs in medicine.