SMYD5 is a ribosomal methyltransferase that trimethylates RPL40 lysine 22 through recognition of a KXY motif

The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here, we identify SET and MYND domain-containing protein 5 (SMYD5) as a ribosomal protein methyltransferase that catalyzes trimethylation of RPL40/eL40 at lysine 22. Through a systematic mass spectrometry-based approach, we identify 12 primary sites of protein methylation in ribosomes from K562 cells, including at RPL40 K22. Through in vitro methylation of synthetic RPL40 using fractionated lysate, we then identify SMYD5 as a candidate RPL40 K22 methyltransferase. We show that recombinant SMYD5 has robust activity toward RPL40 K22 in vitro and that active site mutations ablate this activity. Knockouts of SMYD5 in K562 cells show a complete loss of RPL40 K22 methylation and decreased polysome levels. We show that SMYD5 does not methylate histones in vitro, and by systematic analysis of its recognition motif, we find that SMYD5 requires a KXY motif for methylation, explaining its lack of activity toward histones.