The protein sequences of three known RNA 2′-O-ribose
methylases were used as probes for detecting putative homologs
through iterative searches of genomic databases. We have
identified 45 new positive Open Reading Frames (ORFs),
mostly in prokaryotic genomes. Five complete eukaryotic
ORFs were also detected, among which was a single ORF (YDL112w)
in the yeast Saccharomyces cerevisiae genome.
After genetic depletion of YDL112w, we observed a specific
defect in tRNA ribose methylation, with the complete disappearance
of Gm18 in all tRNAs that naturally contain this modification,
whereas other tRNA ribose methylations and the complex
pattern of rRNA ribose methylations were not affected.
The tRNA G18 methylation defect was suppressed by transformation
of the disrupted strain with a plasmid allowing expression
of YDL112wp. The formation of Gm18 on an in vitro transcript
of a yeast tRNASer naturally containing this
methylation, which was efficiently catalyzed by cell-free
extracts from the wild-type yeast strain, did not occur
with extracts from the disrupted strain. The protein encoded
by the YDL112w ORF, termed Trm3 (tRNA methylation),
is therefore likely to be the tRNA (Gm18) ribose methylase.
In in vitro assays, its activity is strongly dependent
on tRNA architecture. Trm3p, the first putative tRNA ribose
methylase identified in an eukaryotic organism, is considerably
larger than its Escherichia coli functional homolog
spoU (1,436 amino acids vs. 229 amino acids), or any known
or putative prokaryotic RNA ribose methyltransferase. Homologs
found in human (TRP-185 protein), Caenorhabditis elegans
and Arabidopsis thaliana also exhibit a very long
N-terminal extension not related to any protein sequence
in databases.