Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor - PubMed (original) (raw)
Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor
Katelyn A Doxtader et al. Mol Cell. 2018.
Abstract
S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N6-methyladenosine (m6A) methyltransferase METTL16 controls SAM homeostasis by regulating the abundance of SAM synthetase MAT2A mRNA in response to changing intracellular SAM levels. Here we present crystal structures of METTL16 in complex with MAT2A RNA hairpins to uncover critical molecular mechanisms underlying the regulated activity of METTL16. The METTL16-RNA complex structures reveal atomic details of RNA substrates that drive productive methylation by METTL16. In addition, we identify a polypeptide loop in METTL16 near the SAM binding site with an autoregulatory role. We show that mutations that enhance or repress METTL16 activity in vitro correlate with changes in MAT2A mRNA levels in cells. Thus, we demonstrate the structural basis for the specific activity of METTL16 and further suggest the molecular mechanisms by which METTL16 efficiency is tuned to regulate SAM homeostasis.
Keywords: MAT2A; METTL14; METTL16; METTL3; N6-methyladenosine (m6A); S-adenosylmethionine (SAM); SAM homeostasis; metabolism; methyltransferase.
Copyright © 2018 Elsevier Inc. All rights reserved.
Conflict of interest statement
DECLARATION OF INTERESTS
The authors declare no competing interests.
Figures
Figure 1.. Structure of METTL16 Bound to MAT2A Hairpin 1
(A) Domain organization of human METTL16. Crystallization construct (MTD16) is indicated as a blue bar underneath. (B) Organization of the retained intron isoform of MAT2A mRNA. Exons are indicated with blocks. Hairpins 1–6 (hp1–hp6) within the 3′ UTR are shown as schematics. (C) SEC-MALS profile for full-length METTL16. The left axis is the absorbance at 280 nm (blue peak), and the right axis is the measured molecular weight from the scattering data (green line) at each elution volume. The theoretical molecular weight for full-length monomeric METTL16 is 63 kDa, and the average measured molecular weight from MALS data is 58 kDa. (D) Overall structure of MTD16-hp1x in cartoon representation. Protein is shown in blue, RNA in orange, and “clamp” loops interacting with the RNA in cyan. (E) Surface representation colored by the vacuum electrostatic potential of MTD16. RNA is in orange cartoon representation. (F) Close-up view of the catalytic pocket of MTD16, with key side chains shown as blue sticks. Catalytic motif, NPPF (184–187), is labeled with black boxes. Target adenosine for methylation (Ade17) is shown as orange sticks. (G) The catalytic pocket of MTD16-hp1x colored as in (F), aligned with apo MTD16 (PDB: 6B91) shown as pink cartoon. RMSD is 0.4 Å for 181 Cα atoms. The catalytic motif is labeled with black boxes. Conformational difference for Phe187 and Phe188 is indicated with black arrows. (H) The catalytic pocket of MTD16-hp1x colored as in (D), aligned with MTD3 (PDB: 5K7M) shown as green cartoon. RMSD is 4.5 Å over 104 residues. The catalytic motifs for MTD16 (NPPF, 184–187) and MTD3 (DPPW, 395–398) are shown as sticks. Phe188 from MTD16 and Tyr406 from MTD3 are also shown as sticks. Residues are labeled with corresponding colored boxes and text. See also Figure S1.
Figure 2.. Specific RNA Loop Sequence Required for METTL16 Activity
(A) Overall conformation of hp1x in the complex structure with MTD16. Nucleotides are numbered from 5′ to 3′. Target adenosine for methylation is indicated with a red star. Nucleotides in the conserved consensus motif of METTL16 have boxed labels. (B and C) Detailed interactions in the RNA loop region. RNA nucleotides are shown as sticks, color-coded by base identity as in (A). Nucleotides within the consensus sequence are outlined with boxes: UAC (14–16) (B) and G18 (C). Protein residues are shown as gray cartoon and side-chain sticks, labeled with black text. Hydrogen bonds are marked with dashed lines. (D) In vitro methylation activity of METTL16 on wild-type hp1 and hp1 with the indicated loop mutations. Methylated Ade is in red with an asterisk. Mutated base is boldfaced and underlined. Bars correspond to amounts of tritium incorporated into methylated RNA substrates shown as disintegrations per minute (DPM). Data are shown as mean DPM ± SD from three replicates. (E–G) Detailed interactions in the transition region of hp1x. Each panel contains a pair of bases, where (E) contains the pair closest to the loop region, (F) is the next layer of the transition region, and (G) is the final pair, closest to the stem region. RNA bases are shown as sticks, color-coded as in (A). Nucleotides within the consensus sequence are outlined with black boxes. Polypeptide is shown as gray cartoon, with side chains represented with sticks. The ordered water molecule is shown as a red sphere. Hydrogen bonds are shown as dashed lines. (H) In vitro methylation activity of METTL16 on the indicated RNA substrates shown as schematics underneath. Data are shown as mean DPM ± SD from three replicates. See also Figure S2.
Figure 3.. RNA Loop/Transition Features Tune METTL16 Efficiency
(A) In vitro methylation activity of METTL16 on the indicated RNA substrates. Data are shown as mean DPM ± SD from three replicates. (B) Sequence alignment of the human MAT2A 3′ UTR RNA hairpins. Conserved sequences are highlighted in yellow. The key variable regions in the loop and transition areas are indicated with red boxes. (C) Schematics of the RNA substrates used in (D). Hp1 sequences are shown in orange, and hp6 sequences are shown in gray. (D) In vitro methylation activity of METTL16 on the loop/stem chimeras of hp1 and hp6 illustrated with secondary structure schematics in (C). Data are shown as mean DPM ± SD from three replicates. (E) Overall structure of MTD16-hp6 shown as gray cartoon, superimposed on the MTD16-hp1x structure colored as in Figure 1D. RMSD = 0.1 Å for 216 Cα atoms. Longer RNA linker in hp1 versus hp6 is indicated with a black arrow. (F) Close-up view of the structural differences in hp1x RNA backbone (orange) compared to hp6 RNA backbone (gray). R200 and N39 are shown as sticks in blue (MTD16-hp1) and gray (MTD16-hp6) and labeled with black text. The additional two bases in hp1x linker are labeled with black text. (G) In vitro methylation activity of METTL16 on hp1 and hp6. Wild type (WT) METTL16 is compared to full-length METTL16 containing the indicated point mutations. Data are shown as mean DPM ± SD from three replicates. (H) In vitro methylation activity of wild type METTL16 on the wild type hairpins is compared to the effect of RNA mutations at the G-to-A position. Data are shown as mean DPM ± SD from three replicates. (I) In vitro methylation activity of METTL16, wild type or containing R200Q mutation, on hp5. Data are shown as mean DPM ± SD from three replicates. See also Figure S3.
Figure 4.. Autoregulatory K-Loop of METTL16 Blocks the SAM Binding Pocket
(A) Superimposition of MTD16-hp1x (as shown in Figure 1D) onto the structure of MTD16-SAH complex (PDB: 6B92) shown as pink cartoon representation. RMSD is 0.3 Å for 180 Cα atoms. SAH are shown as magenta sticks and labeled with a black arrow. K-loop region is indicated with a black dashed circle. (B) Close-up view of the detailed interactions of the K-loop with the SAM binding pocket. Residues used in mutagenesis studies are shown in stick representation (blue, MTD16-hp1x; pink, MTD16-SAH). Black arrows highlight conformational differences. (C) 2_F_obs -_F_calc map contoured at 1.0 σ for MTD16-hp1x is shown for a portion of the K-loop (blue sticks). (D) In vitro methylation activity of wild-type or mutant METTL16 on hp1. Data are shown as mean DPM ± SD from three replicates. (E) In vitro methylation activity over a range of SAM concentrations, with wild-type or mutant METTL16, for hp1. Data are shown as mean DPM ± SD from three replicates. (F) In vitro methylation activity of wild-type or mutant METTL16 relative to the wild-type activity on the indicated RNA substrates. Data are shown as the ratio of the means, and error is ± the relative SD. See also Figure S4.
Figure 5.. Modulating METTL16 Activity Alters MAT2A mRNA Levels
(A) Schematic of the reporter construct used in cell-based assays. (B) Representative northern blot of β-globin (MAT2A mRNA) in cells transfected with the reporter (A) and wild-type or mutant FLAG-METTL16, from cells grown over a range of methionine concentrations. Images were cropped from the same gel at the same exposure. GAPDH northern blot is shown below as a loading control. (C) Quantification of relative mRNA normalized to_GAPDH_, expressed relative to WT at 1,000 μM methionine from three replicates of (B). Data are shown as the mean ± SD. p values are indicated with asterisks over bars that were compared using unpaired Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001. (D) Representative northern blot of β-globin (MAT2A mRNA) in cells transfected with the reporter (A) and wild-type or mutant FLAG-METTL16, from cells grown over a range of methionine concentrations. Images were cropped from the same gel at the same exposure. GAPDH northern blot is shown below as a loading control. (E) Quantification of relative mRNA normalized to_GAPDH_, expressed relative to WT at 1,000 μM methionine from three replicates of (D). Data are shown as the mean ± SD. p values are indicated with asterisks over bars that were compared using unpaired Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.
Figure 6.. Regulatory Mechanisms of METTL16
(A) In an autoinhibited state, the METTL16 K-loop occludes the SAM binding pocket. This results in less methylation of the MAT2A_3′ UTR, increased splicing, and more stability for the transcript. The increased abundance of MAT2A mRNA results in an increase of SAM biosynthesis. (B) METTL16 can be activated when the K-loop changes conformation to allow for SAM binding. This results in higher methylation efficiency of_MAT2A mRNA, less splicing, and more degradation. The decreased abundance of MAT2A mRNA results in reduced SAM biosynthesis. (C) Mutations tested in this study are marked by effect on in vitro methylation activity: activating (cyan), inhibitory (red), or unaffected (gray). Protein mutations are shown as spheres, and RNA mutations are indicated with outlined boxes.
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