Each transfer RNA (tRNA) is aminoacylated (charged) with a genetic codon-specific amino acid at its 3′ end. Charged tRNAs are primarily used for translation, whereas fluctuations in charged tRNA fractions are known to reflect cellular response to stress. Here we report the development of individual tRNA-acylation using PCR (i-tRAP), a convenient PCR-based method that can specifically quantify individual tRNA charging ratio. In this i-tRAP method, demethylases remove base methylations which are problematic for reverse transcription reaction, and β-elimination reaction specifically removes the 3′ end of adenine residue in uncharged tRNA. Subsequent TaqMan MGB qRT-PCR can distinguish between cDNA of charged tRNA and uncharged tRNA. By using this method, we revealed that the charging ratio of tRNAGln(CUG) was changed in response to amino acid starvation and also the charging ratio of tRNAGln(CUG) in senescent cells was lower than in young cells under starvation conditions. i-tRAP can be applicable to the quantification of charging ratio of various tRNAs, and provides a simple and convenient method for analyzing tRNA charging.

Each tRNA is aminoacylated (charged) with a genetic codon‐specific amino acid. It remains unclear what factors are associated with tRNA charging and how tRNA charging is maintained. By using the individual tRNA acylation PCR method, we found that the charging ratio of tRNAGln(CUG) reflects cellular glutamine level. When uncharged tRNAGln(CUG) increased under amino acid starvation, the kinase GCN2, which is a key stimulator of the integrated stress response, was activated. Activation of GCN2 led to the upregulation of ubiquitin C (UBC) expression. Upregulated UBC, in turn, suppressed the further reduction in tRNAGln(CUG) charging levels. Thus, tRNA charging is sensitive to intracellular nutrient status and is an important initiator of intracellular signaling.