Methyltransferase-like 21C (METTL21C) methylates alanine tRNA synthetase at Lys-943 in muscle tissue.

Protein lysine methylation is a common posttranslational modification (PTM) throughout the human proteome that plays important roles in diverse biological processes. In humans, there are >100 known and candidate protein lysine methyltransferases (PKMTs), many of which are linked to human diseases. Methyltransferase-like protein 21C (METTL21C) is a PKMT implicated in muscle biology that has been reported to methylate valosin-containing protein/p97 (VCP) and heat shock 70kDa protein 8 (HSPA8). However, a clear in vitro methyltransferase activity for METTL21C remains yet to be demonstrated, and whether it is an active enzyme that directly methylates substrate/s in vivo is unclear. Here, we used an unbiased biochemistry-based screening assay coupled to MS, which identified alanine-tRNA-synthetase 1 (AARS1) as a direct substrate of METTL21C. We found that METTL21C catalyzes methylation of Lys-943 of AARS1 (AARS1-K943me) both in vitro and in vivo. In vitro METTL21C-mediated AARS1 methylation was independent of ATP or tRNA molecules. Unlike for AARS1, and in conflict with previous reports, we did not detect METTL21C methylation of VCP and HSPA8. AARS1-K943 methylation in HEK293T cells is dependent upon METTL21C levels. Finally, METTL2C was almost exclusively expressed in muscle tissue, and, accordingly, we detected METTL21C-catalyzed methylation of AARS1 in mouse skeletal muscle tissue. These results reveal that AARS1 is a bona fide in vitro substrate of METTL21C and suggest a role for the METTL21C-AARS1 axis in the regulation of protein synthesis in muscle tissue. Moreover, our study describes a straightforward protocol for elucidating the physiological substrates of poorly characterized or uncharacterized PKMTs.