Fig. 2: A conserved C-terminal arginine residue required for METTL2A/2B/6 tRNA binding and methyltransferase activity.

a HPLC-MS/MS analysis of m³C and m⁵C standards (left). HPLC-MS/MS analysis of indicated tRNAs isolated from WT, METTL2A/2B KO, METTL6 KO, or METTL2A/2B/6 triple KO HEK293T cells (right). b Northern-blot detection of HAC-induced cleavage of tRNA at m³C showing re-expression of METTL2A (M2) but not the METTL2A-R362A mutant (M2-R362A) in METTL2A/B knockout (M2KO) cells can rescue the m³C modification on tRNA-Thr and tRNA-Arg. Blots are representative of repeated experiments. c Multiple sequence alignment of the C terminal end of m³C methyltransferases in different species. The conserved Arginine residue is highlighted in red. Accession numbers are NP_689609.2 (H. sapiens METTL6), NP_014882.4 (S. cerevisiae), NP_596587.2 (S. pombe), NP_001040827.1(C. elegans), NP_647636.3 (D. melanogaster), NP_001308083.1 (H. sapiens METTL8), NP_001017902.1 (D. rerio), NP_766155.3 (M. musculus), NP_859076.3 (H. sapiens METTL2A), NP_060866.2 (H. sapiens METTL2B). d Northern-blot detection of HAC-induced cleavage of tRNA at m³C showing re-expression of METTL6 (M6) but not the METTL6-R259A mutant (M6-R259A) in METTL6 knockout (M6KO) cells can rescue the m³C modification on tRNA-Ser. Blots are representative of repeated experiments. e 3D structure of human METTL6 (PDB: 7f1e) shows the conserved R259 is localized in a positively charged groove next to the SAM binding pocket. f Northern blots of tRNAs interacting with WT and mutant proteins by RNA immunoprecipitation (RIP). M2-R362A and M6-R259A are not able to interact with their tRNA substrates. Blots are representative of repeated experiments. Source data are provided as a source data file.