The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112

Abstract

N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5-TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5-TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.

Figure 1

METTL5 and ZCCHC4 are, respectively, the 18S and 28S rRNA m⁶A methyltransferases. (A) Ribbon representation of the human 40S subunit (11). Ribosomal proteins and 18S rRNA are colored light green and orange, respectively. Helix 44 (h44) of 18S rRNA is highlighted in magenta. The locations of m⁷G₁₆₃₆ and m⁶A₁₈₃₂ rRNA modifications are shown with red and cyan spheres, respectively. A 9-nt mRNA was modeled into the mRNA channel by superimposing the cryo-EM structure of mammalian ribosomal elongation complex bound to eEF1a and tRNAs (74). The mRNA codons corresponding to the A-, P- and E-sites are colored brown, blue and pink, respectively. Inset: Close-up view of the decoding site with some modified nucleotides highlighted. The anticodon loop of the tRNAs bound to the A-, P- and E-sites (delineated by dashed lines) are shown in brown, blue and pink, respectively. The methyl group of the m⁶A₁₈₃₂ nucleotide is shown as a black sphere. The tRNAs were modeled by superimposing the cryo-EM structure of the mammalian ribosomal elongation complex bound to eEF1a and tRNAs (74). (B) Ribbon representation of the human 60S subunit (11). Ribosomal proteins and 28S rRNA are colored light blue and beige, respectively. The respective locations of the m⁶A₄₂₂₀ rRNA modification and peptidyl transferase center (PTC) are shown as green and magenta spheres. CP: Central protuberance. Helix H81 of 28S rRNA is highlighted in red. (C) Human METTL5 is necessary for the formation of m⁶A on 18S but not on 28S rRNA. HPLC chromatograms (A_254nm) of 18S rRNA (top panel) and 28S rRNA (lower panel) purified from mettl5 -/- clones and digested to nucleosides. The digested rRNAs purified from HCT116 parental cells (black curve) were used as controls. (D) Human ZCCHC4 is necessary for the formation of m⁶A on 28S but not on 18S rRNA. HPLC chromatograms (A_{254 nm}) of 18S rRNA (top panel) and 28S rRNA (lower panel) purified from zcchc4 -/- clones and digested to nucleosides. The digested rRNAs purified from HCT116 parental cells (black curve) were used as controls.

Figure 2.

METTL5 and ZCCHC4 are not essential to growth or ribosome biogenesis, and METTL5 gains metabolic stability by associating with human TRMT112. (A) Cell growth is not markedly affected in the absence of METTL5 or ZCCHC4. An identical number of cells of the indicated cell lines was seeded, and cell growth monitored every 24 h for 3 days by cell counting in an hemocytometer after staining with a vital stain. The experiment was performed in triplicate (s.d. shown in gray). (B) Mature rRNA production is not affected in the absence of METTL5 or ZCCHC4. Total RNA extracted from the indicated cell lines was resolved on a denaturing agarose gel and stained with ethidium bromide to reveal the mature 18S and 28S rRNAs. The 28S/18S ratio was extracted from Bioanalyzer electropherograms. (C) Atomic-resolution structure of the human METTL5–TRMT112 complex. The TRMT112 zinc-binding domain (ZBD) and its helical central domain are colored in purple and pink, respectively. The SAM molecule bound to METTL5 is shown as gray sticks. The methyl group transferred from SAM to the RNA substrate upon catalysis is depicted as a black sphere. Secondary structure elements of TRMT112 are labeled in italics. (D) Detailed representations of the METTL5–TRMT112 interface. Residues involved in the interface are shown as sticks. Residues of TRMT112 are labeled in italics. Asterisks (*) indicate residues adopting two alternative conformations according to the 2Fo-Fc electron density map. The top panel shows the hydrophobic core of the interface, while the two other panels show the electrostatic interactions. Hydrogen bonds and salt bridges are depicted by black dashed lines. Same color code as in panel (C). (E) The metabolic stability of METTL5 depends on its association with TRMT112. Western blot analysis of the steady-state accumulation of METTL5 in the presence and absence of TRMT112. Synthesis of Flag-tagged METTL5 was induced by adding tetracycline (see ‘Materials and Methods’ section). TRMT112 synthesis was knocked down with a specific siRNA. Duplicated blots were probed with anti-Flag (METTL5 detection) or anti-WBSCR22 antibodies. As a loading control, the blot was probed with an anti-actin antibody.

Figure 3.

Comparison of the active sites of METTL5–TRMT112 and of an N6-adenosine DNA methyltransferase from Thermus aquaticus, and structure of the m⁶A-methylated area in pre-40S and mature 40S subunits. (A) Superimposition of the METTL5–TRMT112 complex onto the M. TaqI–DNA complex (PDB code: 1G38). The m⁶A protruding into the active site of the M. TaqI methyltransferase is shown as sticks, and the N6 atom, on which the methyl group is grafted, is shown as a blue sphere. The DNA backbone is shown in orange. (B) Zoom-in on the active sites of METTL5 (yellow) and M. TaqI (light brown) (C) Superimposition of mature 40S (rRNA in orange; ribosomal proteins in green and assembly factors in gray; 11) and late pre-40S subunits (rRNA in gray, for the sake of clarity, the proteins and RNA corresponding to the head are not shown; 67). The WBSCR22–TRMT112 complex (blue for WBSCR22 and purple for TRMT112) as observed in state A of late 40S precursors is shown in ribbon representation (PDB code: 6G4W). Helix h44 is highlighted in magenta (mature 40S) and black (immature 40S). The m⁶A₁₈₃₂ as seen in the mature 40S is shown as cyan sticks and the N6 methyl group is shown as a sphere. The location of m⁷G₁₆₃₆ as seen in the cryo-EM structure of the mature ribosome is depicted by a red sphere. (D) Zoom-in on the region centered on position A₁₈₃₂, used to compare RNA conformations within the mature small subunit and late subunit precursors. Same color code as in panel (C).

Figure 4.

Transcriptome-wide mapping of m⁶A by miCLIP reveals that METTL5 and ZCCHC4 are exclusively ribosomal RNA writers. (A) Metagene analysis of all m⁶A sites identified on mRNAs. The called m⁶A sites in each replicate were collapsed and plotted with the MetaPlotR pipeline (75). This shows the typical distribution of m⁶A, with enrichment at the stop codon and a high degree of overlap between the different conditions. DRACH, m⁶A modification consensus motif (D = A, G or U; R = A or G; H = A, C or U). (B) No other sites in the transcriptome are methylated by METTL5 or ZCCHC4. Coverage in sliding windows across each transcript was calculated and normalized per transcript. Differential bins between wild-type and each knockout were tested for significance with edgeR and plotted as a volcano plot. No sites in the transcriptome passed the false discovery rate (FDR) threshold of 0.1, indicating that no mRNA adenosines are methylated by METTL5 or ZCCHC4. (C) The genome track for the 47S pre-rRNA locus confirms that METTL5 and ZCCHC4 are the writers for m⁶A on the 18S and 28S rRNAs, respectively. C-to-T transitions are the most frequent substitution at m⁶A sites caused by antibody cross-linking. As both the 18S and 28S m⁶A marks are in a AAC context (red in the sequence shown below; the m⁶A is indicated by a red dot), reads were filtered for those containing CT to reduce background at this locus. The peaks for the 18S m⁶A and the 28S m⁶A (highlighted) are lost, respectively, in the mettl5- and zcchc4-knockout cells. The miCLIP sequencing data is available on the Gene Expression Omnibus database (accession number GSE128699).

Figure 5.

Tentative positioning of the METTL5–TRMT112 complex within the maturing ribosomal small subunit. (A) Five sequential stages (states ‘A’ to ‘E’) in the late maturation of human 40S subunit established by cryo-EM (67). Orange dot, site of m⁶A modification at the ribosomal decoding site. Only state ‘E’ exhibits in the A₁₈₃₂ area an unassigned density that could be ascribed to METTL5–TRMT112 (see panel B). In states ‘A’, ‘C’ and ‘D’, associated assembly factors (WBSCR22–TRMT112 in state ‘A’; RIOK2 in states ‘C’ and ‘D’) would sterically hinder the binding of METTL5–TRMT112. State ‘B’ shows no extra density in the area of interest. The assembly factors LTV1 (in blue), PNO1 (in green) and ENP1 (in orange), as well as helix 44 (h44, in pink) are shown for reference. PNO1 is detected in all five states while ENP1 is dissociated from pre-40S at the transition between states ‘C’ and ‘D’. The region delimited by the dotted box corresponds to the region shown in panel B. (B) Zoom-in on an unassigned density present in pre-40S state ‘E’ (PDB 6G53; EMDB 4351) in the m⁶A-modified area, and tentative docking of the METTL5–TRMT112 structure. This tentative docking results in SAM being properly positioned with respect to the substrate adenosine. Color scheme as in panel (A). (C) Flag-tagged METTL5 co-purifies with PNO1 but not with ENP1. Expression of Flag-METTL5 was induced in HEK293 cells by addition of tetracycline and complexes containing Flag-METTL5 were captured on anti-Flag beads (see ‘Materials and Methods’ section). Co-precipitated proteins were detected by western blotting using the antibodies indicated. As control, a cell line expressing only the Flag-tag was used.