Lysine methylation is widespread on human proteins, however the enzymes that catalyse its
addition remain largely unknown. This limits our capacity to study the function and regulation
of this modification. Here we report that human METTL21B is a protein methyltransferase,
which methylates lysine 165 of eukaryotic translation elongation factor 1A (eEF1A). The
CRISPR/Cas9 system was used to knock out putative protein methyltransferases METTL21B and
METTL23 in K562 cells. The known eEF1A methyltransferase EEF1AKMT1 was also knocked out
as a control. Targeted mass spectrometry revealed the loss of lysine 165 methylation upon
knock out of METTL21B, and the expected loss of lysine 79 methylation on knock out of
EEF1AKMT1. No loss of eEF1A methylation was seen in the METTL23 knock out. Recombinant
METTL21B was then shown to catalyse methylation on lysine 165 in eEF1A1 and eEF1A2 in
vitro, confirming it as the methyltransferase responsible for this methylation site. METTL21B is
specific to 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 knock outs of the genes encoding putative protein methyltransferases, for the
purpose of 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.