A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop

Abstract

The 3-methylcytidine (m 3 C) modification is widely found in eukaryotic species of tRNA Ser , tRNA Thr , and tRNA Arg ; at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA Ser . Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNA Thr(IGU) from abp140-D strains lacks m 3 C. Curiously, Abp140p is composed of a poorly conserved N-terminal ORF fused by a programed +1 frameshift in budding yeasts to a C-terminal ORF containing an S-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that ABP140 is required for m 3 C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m 3 C and since quantitative analysis shows explicitly that tRNA Thr(IGU) from an abp140-D strain lacks m 3 C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m 3 C methyltransferase activity, which is specific for tRNA Thr(IGU) and not tRNA Phe and occurs specifically at C 32 . We suggest that the C-terminal ORF of Trm140p is necessary and sufficient for activity in vivo and in vitro, based on analysis of constructs deleted for most or all of the N-terminal ORF. We also suggest that m 3 C has a role in translation, since trm140-D trm1-D strains (also lacking m 2,2 G 26 ) are sensitive to low concentrations of cycloheximide.

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Polyamine-regulated +1 ribosomal frameshifting on the OAZ1 mRNA governs the synthesis of antizyme, a regulator of polyamine synthesis in cells (Palanimurugan et al 2004;Kurian et al 2011), and +1 frameshifting is also used to synthesize Est3, a subunit of telomerase (Morris and Lundblad 1997;Taliaferro and Farabaugh 2007), and Abp140 (Trm140), a tRNA methyltransferase (Asakura et al 1998;D'Silva et al 2011;Noma et al 2011). Whereas +1 frameshifting can be triggered when the A site contains a stop codon or a sense codon decoded by a rare tRNA, 21 frameshifting relies on a slippery sequence that allows repairing of the A-and P-site tRNAs with the mRNA and on a downstream secondary structure that impedes forward movement of the ribosome.…”

Factors That Shape Eukaryotic tRNAomes: Processing, Modification and Anticodon–Codon Use

“…In humans i 6 A 37 is found on cytosolic tRNAs Ser and tRNA Ser[Sec] in addition to several mitochondrial tRNAs that contain i 6 A 37 or ms 2 i 6 A 37 among which is the major species tRNA Ser(UGA) [130] that also contains m 3 C 32 (also see [102,131]). Although yeast S. cerevisiae and S. pombe mutants lacking m 3 C 32 exhibit no growth phenotype under various conditions [133,147], the S. cerevisiae double mutant trm140Δ trm1Δ that also lacks m 2 2 G 26 on multiple overlapping tRNAs exhibits slow growth in the presence of the translation inhibitor cycloheximide [147]. …”

Identification of factors that promote biogenesis of tRNACGASer

“…The methylation of C 32 is catalyzed by the Trm140 protein and the modification is known to be present in three out of four tRNA Ser and all three tRNA Thr species [32,33]. Ses1 was found to co-purify with Trm140 and its stimulatory role in the formation of m 3 C was restricted to the serine isoacceptors [31].…”