High Resolution TRNAome Quantification Using Next-generation Sequencing

tRNAs are small non-coding RNA molecules which carry amino acids in protein translation process. tRNAs also play an important role in evolution, disease and protein engineering. tRNAome is the set of all tRNA molecules. Traditional biochemical methods are insufficient for high resolution tRNAome identification and quantification due to high variety of tRNA species, stable secondary structure, high sequence homology, similar physicochemical property and complex nucleotide modifications. In this study, we developed a quantification method for t RNAome based on next-generation sequencing, yielding single-nucleotide resolution and thus distinguishes hundreds of tRNA species. We applied this method on several biological systems. We found that Escherichia coli globally down-regulates its tRNAome in the initial stage of oxidative stress, and restored the tRNA levels when fully adapted. This regulation is a prompt response to oxidative stress. The tRNAome of prokaryotic or eukaryotic expression systems decreases globally when expressing heterologous protein. This is likely to be a similar process as the protection mechanism against viral infection. The tRNAome is tissue-specific in mouse. The majority of predicted tRNA genes are not expressing. There is no correlation between the transcription level of tRNA genes and their adjacent protein-coding genes, indicating the transcriptional regulation system of tRNAs are independent to that of mRNAs, and epigenetic regulation is unlikely to involve. In sum, our new t RNAome sequencing method provides a direct and high resolution experimental technique to investigate translational elongation.