FIG 3.
TRMT1 is necessary for the formation of m2,2G in cytoplasmic and mitochondrial tRNAs. (A) Assay of positive hybridization in the absence (PHA) of G26 modification to monitor m2,2G formation in tRNA. PHA probe spans position G26 between the D-AC stem-loops, while the T-loop probe was used for signal normalization. (B) Northern blot PHA assays with the indicated probes using RNA extracted from control-WT or TRMT1-KO cell lines. (C) Schematic of primer extension assay to monitor the presence of m2,2G at position 26 of tRNA. (D and E) Primer extensions with the indicated nucleus- or mitochondrion-encoded tRNA probes. RT, reverse transcriptase; Um, 2′-O-methyluridine; D, dihydrouridine; m1A, 1-methyladenosine; m1G, 1-methylguanosine; m2G, 2-methylguanosine; ψ, pseudouridine; *, nonspecific signal; >, labeled oligonucleotide used for primer extension.