Abstract
Lysine methylation is widespread on human proteins, however the enzymes that catalyze its addition remain largely unknown. This limits our capacity to study the function and regulation of this modification. Here we used the CRISPR/Cas9 system to knockout putative protein methyltransferases METTL21B and METTL23 in K562 cells, to determine if they methylate elongation factor eEF1A. The known eEF1A methyltransferase EEF1AKMT1 was also knocked out as a control. Targeted mass spectrometry revealed the loss of lysine 165 methylation upon knockout of METTL21B, and the expected loss of lysine 79 methylation on knockout of EEF1AKMT1 No loss of eEF1A methylation was seen in the METTL23 knockout. Recombinant METTL21B was shown in vitro to catalyze methylation on lysine 165 in eEF1A1 and eEF1A2, confirming it as the methyltransferase responsible for this methylation site. Proteomic analysis by SILAC revealed specific upregulation of large ribosomal subunit proteins in the METTL21B knockout, and changes to further processes related to eEF1A function in knockouts of both METTL21B and EEF1AKMT1 This indicates that the methylation of lysine 165 in human eEF1A has a very specific role. METTL21B exists only in vertebrates, with its target lysine showing similar evolutionary conservation. We suggest METTL21B be renamed eEF1A-KMT3. This is the first study to specifically generate CRISPR/Cas9 knockouts of putative protein methyltransferase genes, for substrate discovery and site mapping. Our approach should prove useful for the discovery of further novel methyltransferases, and more generally for the discovery of sites for other protein-modifying enzymes.