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. 2020 Jul 1;295(33):11822–11832. doi: 10.1074/jbc.RA120.014505

Figure 4.

Figure 4.

METTL21C methylates AARS1 in cells. A, METTL21C interacts with AARS1 in cells. Western blotting of Flag-METTL21C IPs with the indicated antibodies. IgG used as a negative control. B and C, AARS1 is methylated by METTL21C in cells. MS quantification of (B) GFP-AARS1 or (C) AARS1 methylation at Lys-943 in 293T cells with or without Flag-METTL21C. LC–MS/MS analysis of purified AARS1 in-gel digested with Glu-C. D, generation of selective AARS1-K943me1/me2/me3 antibodies. Dot blot analysis of ΑARS-K943me1/me2/me3 antibodies reactivity with the indicated serial diluted biotinylated peptides. Blot probed with horseradish peroxidase-conjugated streptavidin (strep-HRP) is shown for equal loading of peptides. E, anti-AARS1-me1/me2 antibodies recognize in vitro METTL21C-methylated recombinant AARS1. In vitro methylation assay as above but with nonradiolabeled SAM. Top panel, Western blotting of reactions with anti-AARS1-K943me1. Middle panel, Western blotting of reaction with anti-AARS1-K943me2. Bottom panel, Coomassie stain of proteins in the reaction. F, AARS1 methylates endogenous AARS1, but not VCP, in cells. Western blots with the indicated antibodies in 293T cells with or without Flag-METTL21C expression. VCPK315me3 is the site of methylation reported to be modified in cells by METTL21C (18) but we do not detect any change upon METTL21C expression. Tubulin is shown as a loading control. G, METTL21C is required for AARS1 methylation in cells. Western blotting analysis with the indicated antibodies of whole cell lysates of Flag-METTL21C stable 293T cells expressing the indicated single guide RNA (control or two independent METTL21C guides). Tubulin is shown as a loading control.