METTL21B Is a Novel Human Lysine Methyltransferase of Translation Elongation Factor 1A: Discovery by CRISPR/Cas9 Knockout
2017; Elsevier BV; Volume: 16; Issue: 12 Linguagem: Inglês
10.1074/mcp.m116.066308
ISSN1535-9484
AutoresJoshua J. Hamey, Beeke Wienert, Kate G.R. Quinlan, Marc R. Wilkins,
Tópico(s)CRISPR and Genetic Engineering
ResumoLysine 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. 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. Protein methylation is emerging as an important regulator of diverse cellular processes, in all kingdoms of life (1.Moore K.E. Gozani O. An unexpected journey: lysine methylation across the proteome.Biochim. Biophys. Acta. 2014; 1839: 1395-1403Crossref PubMed Scopus (74) Google Scholar, 2.Zhang X. Huang Y. Shi X. Emerging roles of lysine methylation on non-histone proteins.Cell Mol. Life Sci. 2015; 72: 4257-4272Crossref PubMed Scopus (88) Google Scholar, 3.Lanouette S. Mongeon V. Figeys D. Couture J.F. 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Protein methyltransferases catalyze the methylation of lysine or arginine residues, and some other amino acids to a lesser degree. Protein lysine methyltransferases can be classified as either SET domain or seven-beta-strand methyltransferases, based upon their catalytic domain (13.Herz H.M. Garruss A. Shilatifard A. SET for life: biochemical activities and biological functions of SET domain-containing proteins.Trends Biochem. Sci. 2013; 38: 621-639Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 14.Falnes P.O. Jakobsson M.E. Davydova E. Ho A. Malecki J. Protein lysine methylation by seven-beta-strand methyltransferases.Biochem. J. 2016; 473: 1995-2009Crossref PubMed Scopus (60) Google Scholar). Although all SET domain methyltransferases specifically methylate lysine residues, seven-beta-strand methyltransferases methylate a wide range of molecules, including proteins, nucleic acids and metabolites (15.Martin J.L. McMillan F.M. 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Eukaryotic elongation factor 1A (eEF1A) 1The abbreviations used are: eEF1A; eukaryotic elongation factor 1A; KMT, lysine methyltransferase; AdoMet, S-adenosyl L-methionine; METTL, methyltransferase like; gRNA, small guide RNA; CRISPR, Clustered regularly interspaced short palindromic repeats; SILAC, stable isotope labelling by amino acids in cell culture. 1The abbreviations used are: eEF1A; eukaryotic elongation factor 1A; KMT, lysine methyltransferase; AdoMet, S-adenosyl L-methionine; METTL, methyltransferase like; gRNA, small guide RNA; CRISPR, Clustered regularly interspaced short palindromic repeats; SILAC, stable isotope labelling by amino acids in cell culture. is a translation factor which delivers amino-acyl tRNA to the ribosome during translational elongation. It is also involved in many other cellular functions, including actin cytoskeleton dynamics, proteasomal degradation and nuclear export (30.Mateyak M.K. Kinzy T.G. eEF1A: thinking outside the ribosome.J. Biol. 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Proteomics. 2016; 15: 164-176Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Knockouts of METTL21B showed a complete loss of lysine 165 methylation, a site for which a methyltransferase had not yet been described. Purified METTL21B could methylate purified eEF1A1 and eEF1A2 in vitro, confirming that METTL21B is the methyltransferase responsible for this site of methylation in both eEF1A isoforms. Proteomic analysis of METTL21B knockout by stable isotope labeling by amino acids in cell culture (SILAC) revealed changes in biological processes and complexes related to eEF1A function, including an upregulation of large ribosomal subunit proteins. In accordance with the recent naming of eEF1A-KMT1 and eEF1A-KMT2, we suggest that METTL21B be renamed as eEF1A-KMT3. Three methyltransferases, EEF1AKMT1, METTL21B and METTL23, were targeted for knockout in K562 cells by CRISPR/Cas9 genome editing, to observe any changes in eEF1A methylation. To minimize the chance that any observed changes were because of off-target effects, two separate small guide RNAs (gRNAs) were used to knockout each methyltransferase. The knockout of EEF1AKMT1, a known eEF1A methyltransferase, served as a proof of concept. The methylation of eEF1A in each knockout was analyzed by targeted mass spectrometry followed by database searches and manual data analysis. For SILAC proteomic analysis of the METTL21B and EEF1AKMT1 knockout cell lines, both gRNA knockouts for each methyltransferase were analyzed with forward (light wild-type and heavy knockout) and reverse (heavy wild-type and light knockout) labeling. This gave four different quantified ratios of each methyltransferase knockout compared with wild-type, accounting for any label or gRNA-specific effects. For CRISPR/Cas9 genome editing a plasmid encoding both the Cas9 protein and the gRNA was used. pSpCas9(BB)-2A-GFP (pX458) was a gift from Feng Zhang (Addgene plasmid #48138) (38.Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (6409) Google Scholar). The Cas9 sequence is coupled to a T2A site and EGFP. Expression of the Cas9 protein results in simultaneous expression of EGFP allowing for selection of positively transfected cells. gRNA sequences were designed using the optimized CRISPR design online tool (http://crispr.mit.edu/) provided by the Zhang lab from Massachusetts Institute of Technology, Boston. For each target gene, two different gRNAs were designed. For primers see supplemental Table S1. K562 cells were maintained in RPMI1640 supplemented with 10% fetal calf serum (FCS) and 1 × penicillin, streptomycin and l-glutamine. Cells were transfected by nucleofection using a Neon Transfection System (Invitrogen, Carlsbad, CA,). Cells (105) were resuspended in nucleofection buffer T and given three pulses of 1450 V for 10 ms. Cells were then cultured for 48–72 h in RPMI1640 supplemented with 10% FCS before fluorescent activated cell sorting. Forty-eight to 72 h after transfection, EGFP positive single cells were sorted into 96-well plates and the plates were left in the incubator for 7–14 days or until small colonies could be seen by the naked eye. Clonal populations were then transferred into one master 96-well plate. Fifty microliters of each well of 70% confluent cultures were transferred into a PCR plate and spun down. Supernatant was discarded by flicking the plate. Each cell pellet was resuspended in 50 μl of QuickExtract™ DNA Extraction Solution (Epicenter, Madison, WI). The plate was heated to 98 °C for 2 min followed by incubation at 65 °C for 6 min. This extract was then used in a PCR of the genomic region that had been targeted for knockout. PCR products were then Sanger-sequenced to identify clones that would result in frameshifts and truncated protein products. Sequence alignment and genomic PCR primer design was carried out using Snapgene software (GSL Biotech LLC, Chicago, IL). For primers see supplemental Table S2. For METTL23 clones, which showed allelic variation of mutations, the allelic genotype of these clones was determined by TA cloning using the pGEM-T Easy cloning system (Promega, Madison, WI). To add adenine overhangs, blunt-ended PCR products from knockout screening were treated with Taq Polymerase. A-tailed PCR products were purified, ligated into the pGEM-T Easy vector and transformed into Escherichia coli. For each clone, 12 successful transformants were picked, plasmids extracted and Sanger-sequenced. K562 cells (wild-type and knockouts) were pelleted, washed once with phosphate-buffered saline, before being resuspended in lysis buffer (50 mm HEPES, 100 mm NaCl, 0.5% v/v Triton X-100, 2 mm DTT, 2 mm EDTA, pH 7.5) and incubated on ice for 30 min. Lysates were clarified by centrifugation (40 min at 14,000 rpm and 4 °C) and separated by SDS-PAGE, after which gel bands were excised, digested and prepared for mass spectrometry, as described previously (21.Hamey J.J. Winter D.L. Yagoub D. Overall C.M. Hart-Smith G. Wilkins M.R. Novel N-terminal and lysine methyltransferases that target translation elongation factor 1A in yeast and human.Mol. Cell. Proteomics. 2016; 15: 164-176Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Samples were analyzed by LC-MS/MS on a Q Exactive Plus (Thermo Fisher Scientific, Waltham, MA), as described previously (21.Hamey J.J. Winter D.L. Yagoub D. Overall C.M. Hart-Smith G. Wilkins M.R. Novel N-terminal and lysine methyltransferases that target translation elongation factor 1A in yeast and human.Mol. Cell. Proteomics. 2016; 15: 164-176Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Raw data were converted to Mascot Generic Format (.mgf) using RawConverter (39.He L. Diedrich J. Chu Y.Y. Yates 3rd., J.R. Extracting accurate precursor information for tandem mass spectra by RawConverter.Anal. Chem. 2015; 87: 11361-11367Crossref PubMed Scopus (162) Google Scholar) (v. 1.0.0.0), with "Experiment Type" set to "Data Dependent" and "Select monoisotopic m/z in DDA" selected. Converted data were then searched against the SwissProt database (2016_03, 550,740 sequences through to 2016_09, 552,259 sequences) using Mascot (v. 2.4, Matrix Sciences) hosted by the Walter and Eliza Hall Institute for Medical Research (Melbourne, Australia) with the following settings: Enzyme: Trypsin (trypsin-digested samples), Asp-N (AspN-digested samples), or LysArginase (LysargiNase-digested samples); Max missed cleavages: 2; Precursor ion tolerance: 4 ppm; Fragment ion tolerance: 20 mmu; Peptide charge: 2+, 3+ and 4+; Instrument: ESI_HCD; Variable modifications: Oxidation (M), Methyl (K), Dimethyl (K), Trimethyl (K) and Methyl (DE). Methyl (N-term), Dimethyl (N-term) and Propyl (N-term) were additionally selected as variable modifications for samples pertaining to N-terminal methylation. For samples from lysates (K562 wild-type and mutants), Taxonomy was set as Homo sapiens (human) and the contaminants database (139 sequences) was additionally selected. For METTL23 knockout samples, the Enzyme was set as Asp-N_ambic, and Max missed cleavages was set as 3. All peptides were identified with expect values <0.05, and spectra were manually annotated to only include fragment ions with 20 ppm error or less. Peptides and their methylation states were analyzed by taking extracted ion chromatograms of the monoisotopic peak (±10 ppm) in Thermo Xcalibur Qual Browser 2.2 SP1.48, as described previously (21.Hamey J.J. Winter D.L. Yagoub D. Overall C.M. Hart-Smith G. Wilkins M.R. Novel N-terminal and lysine methyltransferases that target translation elongation factor 1A in yeast and human.Mol. Cell. Proteomics. 2016; 15: 164-176Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). A tolerance of ±5 ppm was used instead for the eEF1A2 AspN peptide DSTEPAYSEKRY+2 and its methylated states, because of the presence of near-isobaric, co-eluting, non-monoisotopic peaks. Mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE (40.Vizcaino J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2775) Google Scholar) partner repository with the dataset identifier PXD005497. METTL21B was cloned into pET15b for bacterial expression with the Gibson Assembly® Cloning Kit (New England Biolabs, Ipswich, MA), with a C-terminal 6x His tag. The resultant plasmid was confirmed by Sanger sequencing, transformed into E. coli (Rosetta DE3) and METTL21B expression induced with IPTG (1 mm) overnight at 18 °C. eEF1A1 and eEF1A2 were cloned into pD1204-GAL1, with C-terminal 6x His tags, using the Electra® Vector System (DNA2.0, Newark, CA), with the exception that the Sap1 (New England Biolabs) digestion was decoupled from the T4 DNA ligase (New England Biolabs) reaction when cloning eEF1A2, because of the presence of a Sap1 site in eEF1A2 decreasing the efficiency of cloning. Resultant plasmids were confirmed by Sanger sequencing, transformed into wild-type yeast (BY4241) and recombinant proteins expressed according to previous methods (41.Mok J. Im H. Snyder M. Global identification of protein kinase substrates by protein microarray analysis.Nat. Protoc. 2009; 4: 1820-1827Crossref PubMed Scopus (40) Google Scholar), except that induction was overnight at 30 °C. Proteins were purified by immobilized metal affinity chromatography, as described previously (17.Zhang L. Hamey J.J. Hart-Smith G. Erce M.A. Wilkins M.R. Elongation factor methyltransferase 3–a novel eukaryotic lysine methyltransferase.Biochem. Biophys. Res. Commun. 2014; 451: 229-234Crossref PubMed Scopus (14) Google Scholar). Purified eEF1A proteins (10 μm) were incubated with or without purified METTL21B (10 μm) in the presence of S-adenosyl l-methionine (AdoMet, 500 μm) in in vitro methylation buffer (50 mm HEPES-KOH, 20 mm NaCl, 1 mm EDTA, pH 7.4) at 37 °C overnight. Reactions were then resolved by SDS-PAGE and eEF1
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