Identification of the TRM2 gene encoding the tRNA(m⁵U₅₄)methyltransferase of Saccharomyces cerevisiae

Abstract

The presence of 5-methyluridine (m⁵U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m⁵U₅₄)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m⁵U₅₄ nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m⁵U₅₄)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m⁵U₅₄)methyltransferase encoding gene trmA⁺ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m⁵U₅₄)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH₂-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.