ScholarWorks@중앙대학교

초록

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 the 3 ' end of tRNA(Leu) is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3 ' 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, and two other 3 ' tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNA(Leu) concentration but a reduction in both the charged tRNA(Leu) and LeuCAG 3 ' tsRNA, suggesting the 3 ' tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNA(Leu) mutants were also shown to affect the generation of the LeuCAG 3 ' tsRNA further suggesting they are created in a highly regulated process. The fact that the 3 ' tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery. Author summary tRNAs were previously considered only as structural RNAs that read the genetic code from mRNAs to add the appropriate amino acids to the growing polypeptide chain. In recent years, it has been discovered that tRNAs can have non-canonical gene regulatory functions through the production of various types of tRNA-generated small RNAs (tsRNAs). We have studied tsRNAs derived from the 3 ' end of the mature tRNA (3 ' tsRNAs). The 3 ' Leu-CAG-tsRNA promotes the translation of a specific ribosomal protein mRNA by altering mRNA secondary structure, ultimately controlling ribosome biogenesis. This suggests that 3 ' tsRNAs may represent another means of regulating mRNA translation and protein synthesis. Additionally, the 3 ' Leu-CAG-tsRNA was shown to be a useful target for treating a humanized hepatocellular carcinoma model in mice. In the current study, we establish a method showing that the 3 ' tsRNAs are generated from tRNAs that are aminoacylated (attached with an amino acid). Moreover, we found the concentrations of 3 ' tsRNAs generated in a cell are dictated at least in part by the enzymes that conjugate the specific amino acids with cognate tRNAs. This suggests 3 ' tsRNA generation is a highly regulated process and further points to the complexity of this additional layer of post-transcriptional gene regulation.

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