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.