Multisite-specific tRNA:m⁵C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: Identification of the gene and substrate specificity of the enzyme

Abstract

Several genes encoding putative RNA:5-methylcytidine-transferases (m⁵C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m⁵C₉₆₇-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21(DE3). The resulting N-terminally His₆-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni²⁺-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m⁵C-methyltransferase that is capable of methylating cytosine to m⁵C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m⁵C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m⁵C in total yeast tRNA. Moreover no tRNA:m⁵C-methyltransferase activity towards all potential m⁵C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m⁵C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w.