Natural human tRNAAla anticodon variants mistranslate the genetic code

Abstract

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 (tRNAAla) variants with non-synonymous anticodon mutations: tRNAAlaCGC G35T, tRNAAlaUGC G35A, and tRNAAlaAGC C36T. Since alanyl-tRNA synthetase (AlaRS) does not recognize the anticodon, we hypothesized that these tRNAAla variants will mis-incorporate Ala at glutamate (Glu), valine (Val), and threonine (Thr) codons, respectively. We found that expressing the naturally occurring tRNAAla 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 mis-incorporation levels at Glu (0.7%), Val (5%) and Thr (0.1%) codons. Although Ala mis-incorporation was higher at Val codons, cells mis-incorporating Ala at Glu codons had the most severe defect in protein production. The data demonstrate the ability of natural human tRNAAla variants to generate mistranslation leading to defects in protein production that depend on the nature of the amino acid replacement.