Identification of substrates for the skeletal muscle-specific lysine methyltransferases METTL21C and METTL21E

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Protein lysine methylation, catalyzed by S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTases), plays important roles in regulating and fine-tuning protein function. The related mammalian lysine-specific MTases METTL21C and METTL21E are predominantly expressed in skeletal muscle, but their substrates have remained unclear and controversial. Here, we found that METTL21C methylated only a single protein, namely fast myosin-binding protein C (fMyBPC; gene: MYBPC2), in METTL21C-deficient mouse skeletal muscle extracts. METTL21C also showed strong activity on recombinant fMyBPC in vitro, introducing trimethylation at Lys-242. This methylation event was also observed in vivo, and abolished by Mettl21c knockout (KO). The observed in vitro activity of METTL21C on fMyBPC was >100-fold stronger than that observed on AARS1 (alanine-tRNA ligase), a previously reported METTL21C substrate. Interestingly, the two other MyBPC paralogs, which are expressed in cardiac and slow skeletal muscle, were not methylated by METTL21C. For METTL21E, we could not detect in vitro activity. However, we found that monomethylation of Lys-35 (K35) in myosin heavy chain (MYH), a methylation event previously reported in various vertebrate muscles, was abolished by Mettl21e KO in mouse muscle, but not by Mettl21c KO. In human muscles, we detected MYH-K35 methylation at a level similar to that observed in mice, suggesting that the current annotation of human METTL21E as a pseudogene (METTL21EP) may be re-evaluated. Taken together, our results provide new insights into protein methylation in skeletal muscle by identifying fMyBPC as a bona fide muscle-specific substrate of METTL21C, and demonstrating that METTL21E is required for MYH-K35 methylation, thus likely representing the responsible MTase.