i-tRAP (individual tRNA acylation PCR): a convenient method for selective quantification of tRNA charging

Abstract

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.

GCN2 kinase‐mediated upregulation of ubiquitin C maintains intracellular glutamine level and tRNAGln(CUG) charging under amino acid starvation

“…1B), and activated ISR signaling coincided with the reduction in tRNA Gln (GUC) charging ratio and the decrease in intracellular glutamine level. In contrast to amino acid starvation, tRNA Gln (GUC) charging restores quickly upon amino acid supplementation [36]. In line with this, phosphorylated GCN2 was quickly dephosphorylated and expression of ATF4 was suppressed upon amino acid supplementation, followed by upregulation of ATF4 expression (Fig.…”

“…S1B). This sharp decline in cellular glutamine levels specifically corresponded to the decrease in the charging level of tRNA Gln (CUG) [36].…”

“…As a nonessential amino acid, the cellular level of glutamine fluctuates dynamically between consumption and biogenesis in response to nutrients and the external environment. Such cells as mouse embryonic fibroblasts (MEF), renal cell line A498, and TIG-1 show the selective reduction in tRNA Gln charging ratio when they are deprived of extracellular amino acids [9,36]. On the other hand, studies using HEK cells showed the charging ratio of some tRNA isoacceptors decreased after starvation [5].…”

“…On the other hand, the concentration of glutamine continued to decrease afterward, and amino acid starvation for 6 h reduced its concentration to less than 5% of the original concentration. Since it takes 6 h of amino acid starvation to reduce the charging ratio of tRNA Gln (-CUG) [36], it is likely that the charging ratio of tRNA Gln (CUG) cannot be maintained when the glutamine concentration falls below 5% of the initial concentration. Since amino acid concentrations in cells are dynamically maintained between the balance of such biological processes as transport, metabolism, biosynthesis, protein synthesis, and protein degradation [51], a critical change in such biological processes affects the charging ratio of tRNA Gln (CUG).…”

“…We re‐analyzed tRNA sequence data that we generated and published previously (GEO accession number: GSE188862) [36].…”