The 3'tsRNAs are aminoacylated: Implications for their biogenesis

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG tsRNA from 3’ end of tRNA Leu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3’-tsRNAs remains elusive and it is unclear if the 3’-ends of 3’-tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3’charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG 3’-tsRNA is fully charged and originated solely from charged mature tRNA. When the leucyl-tRNA synthetase was knocked down, less tsRNA was generated while the mature tRNA was not reduced further supporting that tsRNA generation is regulated. The fact that the 3’-tsRNA is aminoacylated has implications for their biogenesis and provides additional insights into their biological role in post-transcriptional gene regulation.