METTL10 knockdown reduces the level of lysine 318 methylation in EF1A1 <i>in vivo</i>.

<p>A, HEK293T cells were transfected with either scramble siRNA (Scr) or siRNA against METTL10 (siMETTL10). Twenty-four hours after the transfection, the plasmid for FLAG-EF1A1 was transfected and cultured for an additional 48 hours. Cells were harvested and the METTL10 expression level was analyzed with RT-PCR. n = 3, mean ± SD, **p<0.01. B, FLAG-EF1A1 was purified with anti-FLAG agarose beads from siRNA treated cells. EF1A1 lysine 318 mono-methylation (K318-Me1), di-methylation (K318-Me2), tri-methylation (K318-Me3), and unmethylation (K318-Me0) were analyzed by using a triple stage quadrupole mass spectrometer. The intensity of each peptide was normalized to the quantity of each sample's EF1A1 as determined by the presence of five EF1A1 peptide fragments, and the relative intensity was normalized to Scr of K318-Me0; n = 3, mean ± SD, *p<0.05, **p<0.01. C, <i>In vivo</i> METTL10 knockdown is required for <i>in vitro</i> robust methylation by METTL10. FLAG-EF1A1 purified from Scr or siMETTL10 treated cells was incubated with or without His-METTL10 in the presence of ¹⁴C-labeled SAM. Proteins were separated with SDS-PAGE and stained with Coomassie blue (bottom), the autoradiography was performed using a BAS-5000 image analyzer (top).</p

ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates.

<p>A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).</p

Proteomic identification of substrates for seven-beta-strand MTases.

<p>A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl-l-selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the <i>in vitro</i> reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone.0105394.s003" target="_blank">Table S2</a>.</p

Mammalian METTL10 is an EF1A1 KMT.

<p>A, Phylogenic tree of human KMTs. Proteins were clustered based on DNA sequence by the maximum likelihood method using MEGA version 6 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone.0105394-Tamura1" target="_blank">[45]</a>. The nucleotide sequences used here were as follows: G9a (NCBI ID; NM_006709), GLP (NCBI ID; NM_024757), SUV39H1 (NCBI ID; NM_001282166), ESET (NCBI ID; NM_001145415), DOT1L (NCBI ID; NM_032482), VCPKMT (NCBI ID; NM_024558), CAMKMT (NCBI ID; NM_024766), SMYD2 (NCBI ID; NM_020197), METTL21A (NCBI ID; NM_145280), METTL10 (NCBI ID; NM_212554), and METTL20 (NCBI ID; NM_001135863). B, Schematic structure of human METTL10. A conserved MTase domain, Methyltransf_31 (Pfam ID; PF13847) is located in the middle region. C, Subcellular localization of METTL10. The plasmid for FLAG-tagged METTL10 was transfected into HeLa cells, and the FLAG-tagged proteins were visualized under immunofluorescence microscopy. D, List of METTL10 substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL10 (lane 2 and lane 5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone-0105394-g002" target="_blank">Fig. 2C</a>) and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone-0105394-g002" target="_blank">Figure 2</a>. Molecular weight, peptide area which reflects the protein amount, and fold enrichment of the peptide area are listed. ND, not determined because substrate was detected only in the condition for lane 5 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone-0105394-g002" target="_blank">Figure 2C</a>. The total numbers of identified proteins, 2-fold increase (compared to the control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone.0105394.s004" target="_blank">Table S3</a>.</p

METTL10 tri-methylates K318 in EF1A1.

<p>A, METTL10 methylates EF1A1 <i>in vitro</i>. FLAG-tagged EF1A1 and His-METTL10 were incubated with or without 14C-labeled SAM. Proteins were separated with SDS-PAGE and stained with Coomassie blue (bottom), the autoradiography was detected with the image analyzer BAS-5000 (top). B, A conserved SAM-binding domain is important for MTase activity of METTL10. The MTase domain of METTL10 (WT, 85-DIGTGNG-91) was replaced with alanines (4A, 85-AIATANA-91). The representative gel and its autoradiography was detected as in A. C, The relative EF1A1 methylation was quantified by the intensity of autoradiography, and normalized to the intensity by WT 0 µg as 1 (n = 3, mean ± SD, *p<0.05, **p<0.01). D, MS/MS spectrum of peptide fragments containing trimethylated lysine 318. Box; Asterisks represent b- and y- ions detected. E. Table of the peptide fragment corresponding to amino acids 306-318. Bold numbers represents fragment ions detected in the experiment. F, METTL10 specifically methylates lysine 318. Five lysine methylation sites (lysine 36, lysine 55, lysine 79, lysine 165 and lysine 318) on EF1A1 were substituted with arginine, and their methylations were examined by autoradiography. G, FLAG-EF1A1 (WT and K318R) were incubated with or without His-METTL10 in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-FLAG antibody for loading control (bottom).</p