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

Identification of the gene responsible for m³C modification of yeast tRNA^Thr(IGU). (A) Schematic of tRNA^Thr(IGU) and primer used for assay of m³C₃₂. Modifications found on tRNA^Thr(IGU) are indicated (Weissenbach et al. 1977). The oligonucleotide used for primer extension by reverse transcriptase anneals to residues 55–35. Primer extension of tRNA^Thr(IGU) species that contain m³C₃₂ will terminate at residue 33, whereas species lacking m³C₃₂ will terminate at residue 27 before m^2,2G₂₆. m²G indicates N2-dimethylguanosine; D, dihydrouridine; m^2,2G, N₂, N₂-dimethylguanosine; I, inosine; t⁶A, N6-threonylcarbamoyladenosine; Ψ, pseudouridine; m⁵C, 5-methylcytidine; T, ribothymidine; and m¹A, 1-methyladenosine. (B) Validation of primer extension assay with AlkB-treated tRNA^Thr(IGU). tRNA^Thr(IGU) purified from wild-type yeast cells was treated with the bacterial demethylating enzyme AlkB, and the resultant product was analyzed by the primer extension assay, as described in Materials and Methods. B indicates bulk RNA; P, purified tRNA; FL, full-length; a, 2 μg untreated bulk RNA; b, c, untreated tRNA^Thr(IGU) (0.02 μg and 0.2 μg); d, e, AlkB-treated tRNA^Thr(IGU) (0.02 μg and 0.2 μg); and f, no input RNA. (C) An abp140-Δ strain lacks the m³C primer extension block. Bulk RNA from a set of candidate deletion strains was screened by primer extension. RNA from the abp140-Δ strain lacking ORF YOR239W (lane 15) lacks the m³C primer extension block and has a block corresponding to m^2,2G₂₆. WT indicates RNA from wild-type control strain; AlkB, AlkB treated tRNA^Thr(IGU); and −, no RNA.