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

Trm140p modifies tRNA^Thr(IGU) at position 32. (A) Trm140p does not methylate a C₃₂A variant of tRNA^Thr(IGU). Trm140-ff protein purified from yeast (0.13 μg/μL) was assayed for m³C methyltransferase activity with [α-³²P] CTP-transcribed tRNA^Thr(IGU) (a), tRNA^Thr(IGU) C₃₂A variant (b), or tRNA^Phe (c) for 24 h at 30°C and analyzed as described in Figure 3A. (Lanes d–f) tRNAs treated with buffer controls; (lanes g,h) untreated (g) or DMS-treated (h) tRNA^Phe(GAA), used as p*m³C control; (lane i) Pi control. (B) Modification of unlabeled purified tRNA^Thr(IGU) by Trm140p leads to an m³C₃₂ primer extension block; 0.8 μg tRNA^Thr(IGU) purified from wild-type cells (lane a) or trm140-Δ strains (lanes b,c) was incubated for 24 h at 30°C in methyltransferase assay buffer with 0.13 μg/μL Trm140-ff protein purified from yeast (lanes a,b) or with buffer control (lane c), and the location of the m³C modification was analyzed by primer extension, as described in Figure 2. (Lanes e,f) primer extension of 0.8 μg untreated tRNA^Thr(IGU) from trm140-Δ (e) and wild-type (f) strains; (lane d) no RNA.