Natural human tRNA(Ala) anticodon variants mistranslate the genetic code.

Transfer RNAs (tRNAs) play an essential role in protein synthesis by linking the nucleic acid sequences of gene products to the amino acid sequences of proteins. There are >400 functional tRNA genes in humans, and adding to this diversity, there are many single-nucleotide polymorphisms in tRNAs across our population, including anticodon variants that mistranslate the genetic code. In human genomes, we identified three rare alanine tRNA (tRNA(Ala)) variants with nonsynonymous anticodon mutations: tRNA(Ala) (CGC) G35T, tRNA(Ala) (UGC) G35A, and tRNA(Ala) (AGC) C36T. Since alanyl-tRNA synthetase (AlaRS) does not recognize the anticodon, we hypothesized that these tRNA(Ala) variants will misincorporate Ala at glutamate (Glu), valine (Val), and threonine (Thr) codons, respectively. We found that expressing the naturally occurring tRNA(Ala) variants in human cells led to defects in protein production without a substantial impact on cell growth. Using mass spectrometry, we confirmed and estimated Ala misincorporation levels at Glu (0.7%), Val (5%), and Thr (0.1%) codons. Although Ala misincorporation was higher at Val codons, cells misincorporating Ala at Glu codons had the most severe defect in protein production. The data demonstrate the ability of natural human tRNA(Ala) variants to generate mistranslation, leading to defects in protein production that depend on the nature of the amino acid replacement.