METTL21B Is a Novel Human Lysine Methyltransferase of Translation Elongation Factor 1A: Discovery by CRISPR/Cas9 Knockout

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.

Fig. 1.

EEF1AKMT1 and METTL21B knockout in K562 cells. A, B, Schematic of the EEF1AKMT1 (A) and METTL21B (B) genomic loci. Exons are shown with black boxes and translated regions are indicated by increased box height. Enlarged is the exon that was targeted by CRISPR/Cas9 genome editing; the approximate location and direction of the two small guide RNAs (gRNAs) used for gene knockout are shown with red arrows. C, D, Aligned Sanger sequencing tracks and predicted protein products for clonal K562 cell populations after successful knockout of EEF1AKMT1 (C) and METTL21B (D). Wild-type (WT) K562 cells were transfected with CRISPR/Cas9 plasmids containing one of the two gRNAs targeting EEF1AKMT1 or METTL21B, respectively. Clonal populations were established and successful knockout was determined by genomic PCR followed by sequencing with primers spanning the targeted region. The 20-nucleotide gRNA sequence is shown with a red box and the PAM sequence with a blue box. To ensure knockout on the protein level, clonal populations were chosen that resulted in premature stop codons (*) or frameshift mutations resulting in an altered protein product (indicated by red letters in the protein sequence, with the residues that match the WT protein product shown in black letters).

Fig. 2.

Knockouts of EEF1AKMT1 and METTL21B result in complete loss of methylation at lysines 79 and 165, respectively, in eEF1A1. A, Knockouts of EEF1AKMT1 generated with gRNAs #1 and #2 show a complete loss of mono-, di-, and tri-methylation of lysine 79 in eEF1A1, confirming that it is the sole methyltransferase responsible for this site of methylation. The methylation status of lysine 79 was analyzed by taking mass windows (±10 ppm) corresponding to all relevant methylation states of the eEF1A1 AspN peptide DISLWKFETSKYYVTII⁺³. B, Knockouts of the putative methyltransferase METTL21B generated with gRNAs #1 and #2 show a complete loss of mono-, di-, and tri-methylation of lysine 165 in eEF1A1, indicating that it is the methyltransferase responsible for this site of methylation. The methylation status of lysine 165 was analyzed by taking mass windows (±10 ppm) corresponding to all relevant methylation states of the eEF1A1 tryptic peptide MDSTEPPYSQKR⁺². Peaks were normalized to the most abundant ion for each methylation state. Elution times of peptides are shaded; peaks outside shading are unrelated, near-isobaric ions. me0: unmethylated peptide; me1: monomethylated peptide; me2: dimethylated peptide; me3: trimethylated peptide.

Fig. 3.

eEF1A1 and eEF1A2 are methylated by METTL21B in vitro at lysine 165. Purified human eEF1A1 (A) and eEF1A2 (B) were incubated with or without purified METTL21B in the presence of AdoMet. Both eEF1A1 and eEF1A2 were found to be monomethylated at lysine 165 only when incubated with METTL21B. The methylation status of lysine 165 was analyzed by taking mass windows (±10 ppm) corresponding to all relevant methylation states of the tryptic peptides MDSTEPPYSQKR⁺² (eEF1A1) and MDSTEPAYSEKR⁺³ (eEF1A2). Peaks were normalized to the most abundant ion for each methylation state. Elution times of peptides are shaded; peaks outside shading are unrelated, near-isobaric ions. me0: unmethylated peptide; me1: monomethylated peptide.

Fig. 4.

Ribosomal proteins of the cytosolic large subunit and mitochondrion are differentially expressed upon knockout of METTL21B but not EEF1AKMT1. Box plots showing SILAC ratios (Log₂ fold-change of knockout compared with wild-type) for subsets of proteins in METTL21B or EEF1AKMT1 knockouts compared with wild-type. Intervals show the range of 10–90% of values. p values were determined using a Mann-Whitney test. n.s. indicates p > 0.05, **** indicates p ≤ 0.0001. A, Proteins of the cytosolic large ribosomal subunit (n = 42) have significantly higher SILAC ratios in METTL21B knockout compared with EEF1AKMT1 knockout. B, Proteins of the cytosolic small ribosomal subunit (n = 29) exhibit no change in SILAC ratios between METTL21B knockout and EEF1AKMT1 knockout. C, The mitoribosome (n = 62) is upregulated relative to the other mitochondrial matrix proteins (n = 202) in knockout of METTL21B but not EEF1AKMT1. Additionally, mitochondrial matrix proteins, with mitoribosomal proteins excluded, do not change in SILAC ratios between METTL21B and EEF1AKMT1 knockout. D, Proteins of the mitochondrial large ribosomal subunit (n = 39) have significantly higher SILAC ratios in METTL21B knockout compared with EEF1AKMT1 knockout. E, Proteins of the mitochondrial small ribosomal subunit (n = 23) have significantly higher SILAC ratios in METTL21B knockout compared with EEF1AKMT1 knockout.

Fig. 5.

METTL21B and eEF1A lysine 165 show similar evolutionary conservation. A, Multiple sequence alignment of human METTL21B and its orthologues in Mus musculus (mouse), Oryctolagus cuniculus (rabbit) and Xenopus laevis (frog). Conserved motifs are colored as red (Motif I), blue (Motif Post I), yellow (Motif II) and orange (DXXY motif). B, The crystal structure of METTL21B (residues 24-226) bound to S-adenosyl l-homocysteine (PDB: 4QPN). Motifs are colored as in (A) and S-adenosyl l-homocysteine is colored in cyan. The active site is indicated with an arrow. Visualized in PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC.). C, Multiple sequence alignment of eEF1A from H. sapiens, M. musculus, O. cuniculus, Gallus gallus (chicken), X. laevis, Danio rerio (zebrafish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode) and S. cerevisiae (yeast), showing that lysine 165 is only conserved from frog to human. In species with two eEF1A isoforms, eEF1A1 was used. The equivalent residue to human eEF1A lysine 165 is colored as magenta (lysine), green (alanine) or cyan (serine). Multiple sequence alignments were generated using Clustal Omega (69) with default settings. Asterisks (*) indicate full conservation of a residue, colons (:) indicate highly similar residues and periods (.) indicate moderately similar residues.

Fig. 6.

Structure of eEF1A2 showing known eEF1A lysine methylation sites and their methyltransferases. The crystal structure of eEF1A2 (PDB: 4C0S) showing the six canonical methylation sites (blue) and any known responsible human methyltransferases. Because the N-terminal glycine was not resolved in this structure, the most N-terminal residue (glutamate at position four) is shown instead. Visualized in PyMOL (The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC.).