Skip to main content
. 2022 Jun 8;11:e72780. doi: 10.7554/eLife.72780

Figure 1. ProSeAM-SILAC identifies RPL3 as a substrate of METTL18.

(A) Multiple reaction monitoring (MRM)-based identification of τ-N-methylated histidine in bulk proteins from the indicated cell lines. Data from three replicates (points) and the mean (bar) with SD (error bar) are shown. Significance was determined by Student’s t-test (unpaired, two-sided). (B) Schematic representation of the ProSeAM-SILAC approach. (C) ProSeAM-labeled proteins in cell lysate with recombinant His-METTL18 protein. Biotinylated proteins were detected by streptavidin-HRP. Western blot for α-tubulin was used as a loading control. (D) Venn diagram of proteins identified in two independent ProSeAM-SILAC experiments. The reproducibly detected protein was RPL3. (E) Methylated histidine residue in ectopically expressed RPL3-FLAG was searched by liquid chromatography mass spectrometry (LC-MS/MS). (F) Quantification of methylated and unmethylated peptides (KLPRKTH) from the indicated cells. RPL3-FLAG was ectopically expressed and immunopurified for LC-MS/MS. WT, wild type; MT, Asp193Lys-Gly195Arg-Gly197Arg mutant. (G) MRM-based identification of τ-N-methylhistidine in peptides from RPL3. The τ-N-methylhistidine standard, π-N-methylhistidine standard, and RPL3-FLAG peptide (KLPRKTH) results are shown. MeHis, methylhistidine.

Figure 1—source data 1. Full and unedited blots corresponding to Figure 1C.
Figure 1—source data 2. Primary data for graphs in Figure 1.

Figure 1.

Figure 1—figure supplement 1. Generation of SETD3 and METTL18 knockout (KO) cells.

Figure 1—figure supplement 1.

(A) Chemical structure of histidine, π-N-methylhistidine, and τ-N-methylhistidine. (B) Schematic representation of guide RNAs (gRNAs) designed for CRISPR-Cas9-mediated gene KO. (C) Genomic PCR validated the partial DNA deletion in the METTL18 gene locus. (D, E) Western blot of the indicated proteins to confirm the KO of SETD3 and METTL18 (D) and the quantification (E). α-Tubulin was probed as a loading control and for normalization. (E) Data from three replicates (points) and the mean (bar) with SD (error bar) are shown. (F) Multiple reaction monitoring (MRM)-based identification of π-N-methylated histidine in bulk proteins from the indicated cell lines. Data from three replicates (points) and the mean (bar) with SD (error bar) are shown. MeHis, methylhistidine. (G) Coomassie brilliant blue (CBB) staining of recombinant METTL18 proteins used in this study.
Figure 1—figure supplement 1—source data 1. Full and unedited blots corresponding to Figure 1—figure supplement 1C.
Figure 1—figure supplement 1—source data 2. Full and unedited blots corresponding to Figure 1—figure supplement 1D.
Figure 1—figure supplement 1—source data 3. Full and unedited gel images corresponding to Figure 1—figure supplement 1G.
Figure 1—figure supplement 1—source data 4. Primary data for graphs in Figure 1—figure supplement 1E and F.
Figure 1—figure supplement 2. Characterization of methylhistidine in endogenous RPL3.

Figure 1—figure supplement 2.

(A) Sucrose density gradient for ribosomal complexes. Lysate was prepared with a buffer containing EDTA to dissociate 80S into 40S and 60S. The 60S fraction used for liquid chromatography mass spectrometry (LC-MS/MS) analysis is highlighted in gray. (B) Coomassie brilliant blue (CBB) staining of proteins in the 60S fraction in naïve and METTL18 KO HEK293T cells. (C) Methylated histidine residue in endogenous RPL3 in 60S was searched by LC-MS/MS. (D) Quantification of methylated and unmethylated peptide (KLPRKTH) from endogenous RPL3 in 60S cells.
Figure 1—figure supplement 2—source data 1. Full and unedited gel images corresponding to Figure 1—figure supplement Figure 1—figure supplement 2B.
Figure 1—figure supplement 2—source data 2. Primary data for graphs in Figure 1—figure supplement 2D.