METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis

Abstract

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.

Figure 1.

METTL15 localizes in the mitochondrial matrix. (A) Immunofluorescence labelling of a Flag-tagged METTL15 construct (red) in HeLa cells. Cells were counterstained for the mitochondrial import receptor subunit TOM20 (green) and DAPI (blue). Scale bar, 10 μm. (B) Sub-cellular localisation of METTL15 analysed by western blotting with antibodies against METTL15, Flag, TOM22 (mitochondrial outer membrane), mtSSB1 (mitochondrial matrix) and GAPDH (cytosol). HEK293T cells expressing a Flag-tagged METTL15 construct were fractionated into debris (D, lane 2), cytosol (C, lane3) and mitochondria (M, lane 4–6). ‘T’ indicates the total cell lysate. ‘fl’ indicates full-length TOM22, ‘tr’ stands for truncated TOM22.

Figure 2.

METTL15 is essential for mitochondrial translation. (A) Western blot analysis of METTL15 in wild type (WT) and METTL15 KO HAP1 cells. (B) Mitochondrial de novo protein synthesis was assessed with ³⁵S metabolic labelling. Coomassie blue stained (CBS) gel was used as loading control. (C) Quantification of the band intensities shown in B using ImageJ. (D) Representative example of western blot analysis of NDUFB8, SDHB, UQCRC2, COXII, ATP5A and Beta actin. (E) Quantification of 4 western blot experiments for NDUFB8, SDHB, UQCRC2, COXII, ATP5A. Data were statistically analysed by two-tailed Student's t-test. Error bars represent standard deviation of the mean.

Figure 3.

METTL15 is a m⁴C methyltransferase. Structural Analysis of METTL15. (A) Homology modelling of Homo sapiens METTL15 using E. coli RsmH as a template (PDB: 3TKA). The modelled Homo sapiens structure is superimposed onto the bacterial structure. Grey, E. coli RsmH; green, METTL15 cytidine-binding domain; orange, METTL15 methyltransferase (MTase) domain; red, S-adenosyl methionine; blue, cytidine (B, C) Catalytically important residues responsible for AdoMet-binding. Colour coding is as per (A). Homology modelling was performed using Swiss-Model homology modelling server and visualised using PyMol. (D, E) Comparative quantification of various modified nucleosides in mitochondrially-enriched RNA from WT and METTL15 KO HAP1 cells. Nucleosides were quantified by LC-MS, and are expressed as a percentage of the canonical RNA bases. In (D), the levels of several modified nucleoside are compared between the cell lines, while in (E) the m4C is displayed alone.

Figure 4.

METTL15 methylates position C839 of mitochondrial 12S rRNA. (A, B) Heatmap of targeted BS RNA-Seq reads for the region of the12S rRNA encompassing position C839 and C841 for wild type HAP1 cells (WT) (A) and METTL15 KO HAP1 cells (B), showing cytosines of individual reads (on y-axis). Methylated cytosines are shown in grey, while unmodified cytosines are shown in blue (x-axis). (C) Summary of the targeted BS RNA-Seq results for the C839 and C841 sites in WT and METTL15 KO HAP1 cells. (D) View of the human mtSSU from the interface between mitoribosomal subunits. structure of the mtSSU. Putative localisation of tRNAs (green, yellow, red) are presented from structural alignment of a bacterial ribosome loaded with these RNAs (PDB ID: 5JTE). 12S mt-rRNA is presented as an orange ribbon and MRPSs as grey cartoons. 12S mt-rRNA modifications are localised in the three-dimensional, indicating: the position of the known modifications, m⁵C841, m⁶₂A936 and m⁶₂A937, for which the enzymes responsible, NSUN4 and TFB1M, respectively, were previously characterised (dark grey), the m⁴C839 modification, for which METTL15 was associated in the present study (teal), and m⁵U429, for which the enzyme remains to be identified (light grey). Adapted from Rebelo-Guiomar et al. (45).

Figure 5.

cDNA complementation of METTL15 KO cells.(A) Catalytically important residues, D118, R119 and E297, predicted to bind AdoMet (red), that were subjected to site-directed mutagenesis are indicated on the homology model as per Figure 1. The dotted line represents the predicted interactions with AdoMet based on Wei et al. (2012). (B) Western blot analysis of METTL15, NDUFB8 (complex I), SDHB (complex II), UQCRC2 (complex II), COXII (complex IV), ATP5A (complex V) and Beta actin in wild type HAP1 cells, METTL15 KO cells, METTL15 KO cells transfected with cDNA encoding E297A or D118A + R119A mutants or with WT METTL15 cDNA. CBS stands for Coomassie blue staining. (C) Quantification of NDUFB8 analysed by western blot (n = 3, data were statistically analysed by two-tailed Student's t-test. Error bars represent standard deviation of the mean). (D) Pie charts of BS RNA-Seq results showing methylation percentage of 12S mt-rRNA position C839 and C841 in wild type HAP1 cells, METTL15 KO cells, and METTL15 KO cells complemented with E297A, D118A + R119A mutants or with WT METTL15 cDNA.

Figure 6.

Effect of METTL15 inactivation on the mitochondrial ribosome. (A) An average (n = 4) proteomic profile of mt-SSU, mt-LSU and METTL15 upon sucrose gradient sedimentation. (B) Immunopurification of components of the mitochondrial ribosome using a flag-tagged version of METTL15. HEK293T cells expressing a flag-tagged METTL15 construct were lysed and immunoprecipitation was performed using anti-Flag antibodies. The lysate (Input) and eluates were analysed by western blot with antibodies as indicated. (C–F) A representative quantitative sucrose gradient sedimentation analysed by mass spectrometry. Wild type and METTL15 KO HAP1 cells were grown in heavy or light labelled medium and the different fractions after sucrose gradient sedimentation were analysed by mass spectrometry. Graphical representation of total mt-SSU proteins (C) and cytoplasmic SSU proteins (D) or mt-LSU proteins (E) and cytoplasmic LSU proteins (F) detected in each fraction for wild type (grey) or METTL15 KO HAP1 (blue) cells. See also Supplementary Dataset. (G) Western blot analysis of three independent wild type and METTL15 KO HAP1 samples for uS17m, uL3m and Beta-actin. CBS stands for Coomassie blue staining. Asterisk represents non-specific bands. (H) Quantification of band intensities shown in G (n = 3, data were statistically analysed by two-tailed Student's t-test. Error bars represent standard deviation of the mean). (I) Protein steady-state levels of the mt-SSU components in METTL15 KO HAP1 cells, relative to wild type control cells, in the mt-SSU fractions. The most affected proteins are coloured. The results were obtained from duplicate experiments with reciprocal SILAC (Control:heavy/METTL15 KO:light or Control:light/METTL15 KO:heavy) (J) Representative example of relative intensity profiles of the mt-SSU proteins detected in METTL15 KO HAP1 that were in the bottom 20% of steady state level values, as compared to the WT control.

Figure 7.

Possible role of methylation of position C839 of mitochondrial 12S rRNA. (A) Structure of mt-SSU (PDB: 5AJ4) (4) indicating the proteins that are downregulated in METTL15 KO as compared to WT samples. uS15m in dark blue, uS17m in light blue, mS38 in red and uS12m in orange. (B) Detail of (A) showing the localisation of mS38 (red) and mt-rRNA positions C839 and C841. (C) Detail of (A) showing the localisation of uS12m (orange) and mt-rRNA positions C839 and C841. (D) Model for the role of METTL15 in mt-SSU assembly. (i) Partially assembled mt-SSU. (ii) METTL15 methylates 12S mt-rRNA at C839. Teal sphere denotes the methyl group. (iii) Methylation of C839 introduces a structural change on 12S mt-tRNA and enables the incorporation of late stage ribosomal components into mt-SSU.