<p>A, METTL10 methylates EF1A1 <i>in vitro</i>. FLAG-tagged EF1A1 and His-METTL10 were incubated with or without 14C-labeled SAM. Proteins were separated with SDS-PAGE and stained with Coomassie blue (bottom), the autoradiography was detected with the image analyzer BAS-5000 (top). B, A conserved SAM-binding domain is important for MTase activity of METTL10. The MTase domain of METTL10 (WT, 85-DIGTGNG-91) was replaced with alanines (4A, 85-AIATANA-91). The representative gel and its autoradiography was detected as in A. C, The relative EF1A1 methylation was quantified by the intensity of autoradiography, and normalized to the intensity by WT 0 µg as 1 (n = 3, mean ± SD, *p<0.05, **p<0.01). D, MS/MS spectrum of peptide fragments containing trimethylated lysine 318. Box; Asterisks represent b- and y- ions detected. E. Table of the peptide fragment corresponding to amino acids 306-318. Bold numbers represents fragment ions detected in the experiment. F, METTL10 specifically methylates lysine 318. Five lysine methylation sites (lysine 36, lysine 55, lysine 79, lysine 165 and lysine 318) on EF1A1 were substituted with arginine, and their methylations were examined by autoradiography. G, FLAG-EF1A1 (WT and K318R) were incubated with or without His-METTL10 in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-FLAG antibody for loading control (bottom).</p
