RESUMO
To maintain optimal proteome, both codon choice of each mRNA and supply of aminoacyl-tRNAs are two principal factors in translation. Recent reports have revealed that the amounts of tRNAs in cells are more dynamic than we had expected. High-throughput methods such as RNA-Seq and microarrays are versatile for comprehensive detection of changes in individual tRNA amounts, but they suffer from inability to assess signal production efficiencies of individual tRNA species. Thus, they are not the perfect choice to measure absolute amounts of tRNAs. Here, we introduce a novel method for this purpose, termed Oligonucleotide-directed Three-prime Terminal Extension of RNA (OTTER), which employs fluorescence-labeling at the 3'-terminus of a tRNA by optimized reverse primer extension and an assessment step of each labeling efficiency by northern blotting. Using this method, we quantified the absolute amounts of the 34 individual and 4 pairs of isoacceptor tRNAs out of the total 42 nuclear-encoded isoacceptors in the yeast Saccharomyces cerevisiae. We found that the amounts of tRNAs in log phase yeast cells grown in a rich glucose medium range from 0.030 to 0.73 pmol/µg RNA. The tRNA amounts seem to be altered at the isoacceptor level by a few folds in response to physiological growing conditions. The data obtained by OTTER are poorly correlated with those by simple RNA-Seq, marginally with those by microarrays and by microscale thermophoresis. However, the OTTER data showed good agreement with the data obtained by 2D-gel analysis of in vivo radiolabeled RNAs. Thus, OTTER is a suitable method for quantifying absolute amounts of tRNAs at the level of isoacceptor resolution.
