Author response: Structural insights into the molecular mechanism of the m6A writer complex (2016)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Methylation of adenosines at the N6 position (m6A) is a dynamic and abundant epitranscriptomic mark that regulates critical aspects of eukaryotic RNA metabolism in numerous biological processes. The RNA methyltransferases METTL3 and METTL14 are components of a multisubunit m6A writer complex whose enzymatic activity is substantially higher than the activities of METTL3 or METTL14 alone. The molecular mechanism underpinning this synergistic effect is poorly understood. Here we report the crystal structure of the catalytic core of the human m6A writer complex comprising METTL3 and METTL14. The structure reveals the heterodimeric architecture of the complex and donor substrate binding by METTL3. Structure-guided mutagenesis indicates that METTL3 is the catalytic subunit of the complex, whereas METTL14 has a degenerate active site and plays non-catalytic roles in maintaining complex integrity and substrate RNA binding. These studies illuminate the molecular mechanism and evolutionary history of eukaryotic m6A modification in post-transcriptional genome regulation. https://doi.org/10.7554/eLife.18434.001 Introduction Methylation of the amino group at the N6 position in adenine nucleobases (m6A) has emerged as a dynamic posttranscriptional RNA modification (Fu et al., 2014; Liu and Pan, 2016; Meyer and Jaffrey, 2014; Yue et al., 2015). As the most abundant internal modification in eukaryotic messenger and long non-coding RNAs (Dominissini et al., 2013; Meyer et al., 2012), m6A has been shown to regulate the processing (Alarcón et al., 2015b; Xiao et al., 2016), translation (Lin et al., 2016; Wang et al., 2015) and stability (Wang et al., 2014a) of cellular transcripts. In mRNAs, the modification is typically found clustered around the stop codon and within 3’ untranslated regions (Dominissini et al., 2012; Meyer et al., 2012); the presence of m6A in 5’ untranslated regions has in turn been shown to promote 5’ cap-independent translation (Meyer et al., 2015). m6A is implicated in controlling stem cell differentiation and embryonic development (Batista et al., 2014; Chen et al., 2015; Geula et al., 2015; Wang et al., 2014b), the mammalian circadian clock (Fustin et al., 2013), as well as in stress responses such as heat shock (Zhou et al., 2015). The modification is reversible due to the interplay of methyltransferase (MTase) ‘writer’ (Liu et al., 2014) and demethylase ‘eraser’ (Jia et al., 2011; Zheng et al., 2013) enzymes. Transcripts containing m6A marks are specifically recognized and regulated by ‘reader’ proteins, in particular members of the YTH domain family (Stoilov et al., 2002). The cytoplasmic protein YTHDF2 specifically recognizes and destabilizes m6A-modified RNAs by promoting their localization to processing (P-) bodies (Wang et al., 2014a), while YTHDF1 stimulates mRNA translation by interactions with translation initiation factors (Wang et al., 2015). The nuclear YTHDC1 protein modulates pre-mRNA splicing by recruiting and modulating splicing factors (Xiao et al., 2016). Another m6A reader, HNRNPA2B1, has been shown to promote pri-microRNA processing through interactions with the Microprocessor complex (Alarcón et al., 2015a). The m6A RNA modification is catalyzed in the nucleus by a multiprotein writer complex containing the putative methyltransferases METTL3 and METTL14 (Bokar et al., 1997; Liu et al., 2014; Schwartz et al., 2014). The two subunits interact directly with each other and further associate with Wilms tumor 1-associated protein (WTAP) (Liu et al., 2014; Ping et al., 2014; Schwartz et al., 2014). WTAP does not have MTase activity but is required for efficient RNA methylation in vivo and for localization of METTL3 and METTL14 to nuclear speckles (Ping et al., 2014). The m6A writer complex catalyzes the transfer of a methyl group from the donor substrate S-adenosyl methionine (SAM) to the adenine nucleobases in acceptor RNA substrates (Bokar et al., 1997). The complex preferentially modifies substrate RNAs conforming to the consensus sequence GGACU but otherwise appears to have little specificity for structural features in its RNA substrates (Liu et al., 2014). Both METTL3 and METTL14 have been shown to have weak m6A methyltransferase activity in isolation. However, physical interaction between METTL3 and METTL14 results in substantially higher MTase activity than the sum of the activities of the individual methyltransferase subunits (Liu et al., 2014). Despite recent advances in understanding the biological roles of m6A modification, the molecular architecture of the m6A writer complex and its mechanism of RNA methylation are poorly understood. In particular, the molecular basis for the synergistic enhancement of MTase activity within the m6A writer complex is unknown. To shed light on the molecular mechanism of m6A RNA methylation, we determined crystal structures of the catalytic core of the m6A writer complex comprising METTL3 and METTL14 methyltransferase subunits. The structures reveal the molecular architecture of the complex and highlight specific features involved in complex assembly, substrate recognition and catalysis. Crucially, our structural and biochemical data imply that METTL3 is the sole catalytic subunit of the complex, while METTL14 plays non-catalytic roles in complex stabilization and substrate RNA recruitment. These findings advance our mechanistic understanding of the m6A modification in post-transcriptional genome regulation. Results Identification and structure determination of a minimal core m6A writer complex The m6A writer complex contains two putative catalytic subunits - METTL3 and METTL14. Previous studies have shown that METTL3 and METTL14 physically interact with each other and that their association has a synergistic effect on the catalytic activity of the complex (Liu et al., 2014). To obtain structural insights into the molecular architecture and mechanism of the m6A writer complex, we sought to determine the crystal structure of the catalytic core of the complex comprising the two methyltransferase (MTase) subunits (Figure 1A). To this end, we co-expressed human METTL3 and METTL14 in insect cells. The purified METTL3-METTL14 complex was catalytically active (Figure 1B) but failed to crystallize. Both METTL3 and METTL14 polypeptides contain a central MTA-70 methyltransferase domain (Bujnicki et al., 2002). METTL3 additionally contains an N-terminal region harboring two putative CCCH zinc finger motifs, whereas in METTL14, the MTA-70 domain is flanked by N- and C-terminal extensions containing low-complexity sequences (Iyer et al., 2016) (Figure 1A). To identify protein constructs amenable to crystallization, the full-length METTL3-METTL14 complex was subjected to limited proteolysis with chymotrypsin and N-terminal sequencing. Based on these experiments, we engineered N- and C-terminally truncated forms of both METTL3 and METTL14, comprising residues 354–580 and 107–395, respectively. The MTase activity of the truncated METTL3-METTL14 dimer was reduced to background level, indicating that the terminal extensions are required for efficient substrate RNA binding or catalysis. Further experiments using complexes in which only METTL3 or METTL14 was truncated showed that the catalytic activity is contingent upon the presence of full-length METTL3, while the N- and C-terminal extensions flanking the central region comprising residues 107–395 in METTL14 are dispensable (Figure 1B). The truncated METTL3-METTL14 complex was crystallized and its structure solved by a single-wavelength anomalous diffraction approach utilizing the anomalous signal of native sulfur atoms. Experimentally-phased electron density maps revealed the presence of a single molecule of the donor product S-adenosyl homocysteine (SAH), serendipitously co-purified with the complex, in the METTL3 active site (Figure 1—figure supplement 1A). High-resolution native data were subsequently measured from a METTL3-METTL14 crystal grown in the presence of SAH and a short m6A-containing oligonucleotide. However, the RNA oligonucleotide was not evident in electron density maps. The final atomic model, containing a single METTL3-METTL14 dimer in the asymmetric unit, was refined at a resolution of 1.85 Å with an Rfree of 19.0%, working R of 15.7%, and good stereochemistry (Table 1). We additionally co-crystallized the METTL3-METTL14 core complex in the presence of the donor substrate S-adenosyl methionine (SAM) (Figure 1—figure supplement 1B). Using these crystals, a structure of the METTL3-METTL14 complex, in which the co-purified SAH product was exchanged by SAM, was determined at a resolution of 1.9 Å and refined to an Rfree and Rwork values of 18.6% and 15.7%, respectively. No additional electron density attributable to SAM or SAH could be observed in the proximity of METTL14 in the two structures. Disulfide bridge formation was observed between Cys338METTL14 and Cys388METTL14 (Figure 1—figure supplement 2A). The MTase activity of the METTL3-14 complex has previously been reported to be sensitive to reducing agents (Li et al., 2016), suggesting that disulfide bond formation could play a role in regulating the catalytic activity of the complex. However, substitution of Cys338METTL14 with alanine had little impact on MTase activity, indicating that disulfide bond formation in METTL14 is not required for the function of the m6A writer complex (Figure 1—figure supplement 2B). Figure 1 with 2 supplements see all Download asset Open asset Domain composition and methyltransferase activity of METTL3-METTL14. (A) Domain composition of METTL3 and METTL14. Predicted CCCH zinc finger motifs in the N-terminal region of METTL3 are shown in green. The crystallized METTL3-METTL14 complex contained the MTA-70 methyltransferase domain of METTL3 (residues 354–580, highlighted in blue) and a METTL14 construct (residues 117–395, highlighted in pink) comprising the MTA-70 domain preceded by an N-terminal extension (denoted HM). (B) Methyltransferase activities of the full-length METTL3-METTL14 complex, and complexes containing truncated METTL3 and METTL14 proteins used for the structure determination. The activities were determined by measuring the concentration of methylated RNA product using a m6A-specific antibody in an enzyme-linked immunosorbent assay. The activities are represented as fractions of the activity of WT complex. Reactions lacking the donor substrate SAM were performed to determine background signal in the assay due to non-specific antibody binding. Error bars represent mean ± standard error of the mean (s.e.m) of three independent replicates. https://doi.org/10.7554/eLife.18434.002 Table 1 Data collection and refinement statistics. https://doi.org/10.7554/eLife.18434.005 SAM complexSAH complexSulfur-SADX-ray sourceSLS X06DA (PXIII)SLS X06DA (PXIII)SLS X06DA (PXIII)Space groupP 32 2 1P 32 2 1P 32 2 1Cell dimensionsa, b, c (Å)63.95, 63.95, 225.8663.82, 63.82, 225.6364.02, 64.02, 226.84α, β, γ (°)90.0, 90.0, 120.090.0, 90.0, 120.090.0, 90.0, 120.0Wavelength (Å)1.039651.039652.07334Resolution (Å)(2.02–1.90)(1.96–1.85)(2.80–2.70)Rsym (%)8.9 (70.6)7.5 (68.9)16.5 (97.3)CC1/299.9 (89.0)100.0 (87.5)100.0 (97.9)I / σI21.89 (3.42)24.51 (3.55)47.24 (8.03)Observations846792 (131654)906619 (142053)3073142 (279442)Unique reflections81250 (13087)87473 (14080)28625 (2970)Multiplicity10.4 (10.1)10.4 (10.1)107.4 (94.1)Completeness (%)99.9 (99.2)99.9 (99.3)100.0 (100.0)RefinementResolution (Å)44.62–1.9049.65–1.85No. reflections4327146510Rwork/Rfree0.1567/0.18550.1569/0.1899No. atomsProtein35493571Ligands3231Water359405B-factorsMean28.7629.89Protein28.1729.08Ligands25.0622.72Water35.2837.64R.m.s. deviationsBond lengths (Å)0.0110.011Bond angles (°)1.0881.126Ramachandran plot% Favored98.198.8% Allowed1.91.2% Outliers00 Values in parentheses denote highest resolution shell. METTL3 and METTL14 form a pseudosymmetric dimer The structures of the METTL3-METTL14 complex reveal that the two methyltransferase subunits form a compact pseudosymmetric heterodimer with overall dimensions of 80 × 40 × 35 Å (Figure 2A). Both METTL3 and METTL14 belong to the Group I clade of eukaryotic N6A-MTases (Iyer et al., 2016). The core of each methyltransferase subunit is formed by a Rossman fold domain comprising a central, curved, eight-stranded beta sheet flanked by four alpha helices (Figure 2B, Figure 2—figure supplement 1). The METTL3 and METTL14 MTase domains dimerize through an extensive interface that buries 2055 Å2 of solvent-accessible surface area per subunit. The symmetric part of the dimerization interface is formed by strands β4–5 and helix α4 of each MTase domain (Figure 2C), and involves a network of hydrogen bonding interactions as well as hydrophobic contacts (Figure 2D). The hydrophobic region of the METT3-METTL14 interface, centered on strands β4–5, is capped by the side chain of Ile134METTL14 from an N-terminal extension of METTL14 comprising residues Asn117-Asp136METTL14 (Figure 1A). The METTL14 N-terminal extension, which lacks secondary structure except for a short alpha-helical motif (Tyr119-Thr126METTL14), breaks the overall pseudo-C2 symmetry of the heterodimer and shields the hydrophobic dimerization interface from solvent exposure. Of note, we were unable to express soluble METTL3-METTL14 complexes lacking the N-terminal METTL14 extension. Unlike METTL14, METTL3 lacks equivalent amino acid residues in the region upstream of the methyltransferase domain. Finally, the dimerization interaction is reinforced by two partially disordered loops, one from each subunit (residues Arg468-Lys480METTL3 and Lys266-Arg283METTL14), that reach over and insert an aromatic residue (Trp475METTL3 and Phe281METTL14, respectively) into a hydrophobic pocket in the other subunit (Figure 2E). Figure 2 with 2 supplements see all Download asset Open asset METTL3 and METTL14 form an asymmetric dimer. (A) Overall structure of METTL3-METTL14 heterodimer. METT3 is colored blue; METTL14 is colored pink. The color coding is used throughout the manuscript. Bound SAM donor substrate is shown in stick format and colored yellow. (B) Sequence alignment of human METLL3 and METTL14. The alignment was generated using T-Coffee (Di Tommaso et al., 2011) and displayed using Espript (Gouet et al., 1999). Invariant residues are highlighted in red; conserved residues are highlighted in yellow. Amino acid residues involved in donor or acceptor substrate binding are marked with colored triangles. Secondary structure elements in METTL3 are depicted above the aligned amino acid sequences. (C) Enlarged view of the dimerization interface (orientation as in the left panel of A). (D) N-terminal helical motif of METTL14 shields the hydrophobic part of the METTL3-METTL14 interface from the solvent exposure by capping it with Ile134Mettl14. (E) Detailed views of intersubunit interactions mediated by loops Arg468-Lys480METTL3 and Lys266-Arg283METTL14 containing hydrophobic residues Trp475-Leu476METTL3 and Val280-Phe281METTL14, respectively. https://doi.org/10.7554/eLife.18434.006 The structure of the human METTL3-METTL14 complex has been recently reported by two other studies (Wang et al., 2016a, 2016b). Both studies utilized a slightly different METTL3-METTL14 construct, yielding a different crystal form of the complex. Overall, all structures are in good agreement with one another, the major difference being the absence of an ordered polypeptide chain linking the N-terminal helical motif of METTL14 and the methyltransferase domain in the structure reported in this study (Figure 2—figure supplement 2). Additionally, the structure reported in this study lacks a C-terminal helix in METTL14 due to a different choice of METTL14 domain boundaries and instead contains the Cys338METTL14–Cys388METTL14 disulfide bridge. Further structural differences between the different crystal forms can be attributed to the intrinsic flexibility of loop regions and differences in crystal packing (Figure 2—figure supplement 2). Individually, the MTase domains of METTL3 and METTL14 are highly similar to other Rossman fold-containing methyltransferases. The closest structural homologs include bacterial adenine-specific DNA methyltransferases MboIIa (Osipiuk et al., 2003) (rmsd of 2.8 Å over 167 Cα atoms for METTL3, 2.7 Å over 165 Cα atoms for METTL14) and RsrI (Scavetta et al., 2000) (rmsd of 2.8 Å over 172 Cα atoms for METTL3, 2.8 Å over 171 Cα atoms for METTL14). However, the dimerization mode observed in the METTL3-METTL14 complex is distinct from these other multisubunit RNA methyltransferase enzymes such as Bud23-Trm12 (Létoquart et al., 2014) or Trm9-Trm112 (Létoquart et al., 2015) and closely resembles the homodimerization mode of prokaryotic N6A DNA methyltransferases such as EcoP15I (Figure 3) (Gupta et al., 2015). Figure 3 Download asset Open asset Dimerization of METTL3-METTL14 is similar to homodimeric N6-adenosine DNA methyltransferases. (A) Structure of dimeric N6-adenosine DNA methyltransferase EcoP15I bound to a DNA substrate (Gupta et al., 2015). (B) Inset showing detail of the dimer interface of EcoP15I (colored gray) superimposed with the METTL3-METTL14 (METTL3 colored in blue, METTL14 in pink). https://doi.org/10.7554/eLife.18434.009 Donor and acceptor substrate binding in the METTL3 active site The donor substrate binding site in METTL3 is located at the carboxy-termini of strands β1, β7 and β8, where the SAM donor is bound by multiple hydrogen bonding interactions (Figure 4A). The SAM binding site is enclosed from opposite sides by partially disordered loop containing Asp395-Thr408METTL3 (termed active site loop 1, ASL1) and a fully ordered loop comprising residues Arg508-Lys513METTL3 (hereafter termed active site loop 2, ASL2). A conserved DPPW motif (Asp395-Pro398METTL3) in ASL1, characteristic of N6-adenosine methyltransferases (Iyer et al., 2016), is likely involved in coordinating the adenine group of the acceptor substrate. The methyl group of SAM is positioned directly above the DPPW motif, while the adenine moiety is inserted in a hydrophobic pocket lined with Ile378METTL3, Pro397METTL3, Leu409METTL3 and Phe534METTL3, and is coordinated by hydrogen binding interactions to Asp377METTL3 and the main-chain amide nitrogen of Ile378METTL3. The ribose 2’ and 3’ hydroxyl groups are hydrogen bonded to Asn549METTL3 and Gln550METTL3, respectively. The amino group of the methionine moiety is hydrogen bonded to Arg536, while the carboxyl group is surrounded by the side chains of His512METTL3, Lys513METTL3, Arg536METTL3, and His538METTL3. Comparisons with the structure of the SAH product-bound complex reveal that the both SAM and SAH molecules adopt very similar conformations except for a slight rotation of the ribose moiety in SAH (Figure 4B). Additional ordering of ASL1 is observed in the SAH-bound structure, whereby Tyr406METTL3 caps the SAH product and makes a hydrogen bonding interaction with Ser511METTL3 in ASL2. Taken together, the structures of the SAM- and SAH-bound METTL3-14 complexes thus reveal the METTL3 active site in the pre- and post-catalytic states, respectively. Figure 4 Download asset Open asset Methyl group donor substrate and product binding in the METTL3 active site. The active site of METTL3 with (A) the donor substrate SAM and (B) product SAH bound. The ligands are shown in stick format and colored yellow. Black dashed lines indicate hydrogen bonding interactions. (C) Model of acceptor adenine binding in the METTL3 active site. Donor (SAM) and acceptor (adenine) substrates are shown in stick format and colored gray. The model was generated by superimposing METTL3 with the structure of N6-adenosine DNA methyltransferases EcoP15I (Gupta et al., 2015). https://doi.org/10.7554/eLife.18434.010 Superposition of METTL3 onto the structure of other methyltransferase-substrate complexes such as EcoP15I suggests a mechanism for acceptor substrate recognition, in which the adenine moiety of the acceptor substrate is inserted between ASL1 and ASL2 (Figure 4C). The N6 amino group is located such that it is hydrogen-bonded to the side chain amide group of Asp395METTL3, the hydroxyl group of Tyr406METTL3 and the main chain carbonyl of Pro396METTL3 within the conserved DPPW motif. These interactions activate the amino group for nucleophilic attack on the methyl group in SAM. Additionally, a rearrangement of the ASL1, such that Trp398METTL3 forms a π-π stacking interaction with the acceptor adenine, would be expected to occur upon acceptor substrate binding. METTL14 contains a degenerate active site and lacks catalytic activity In comparison with METTL3, the MTase domain of METTL14 adopts a very similar fold, superimposing with an rmsd of 1.65 Å over 199 aligned residues (Figure 2B, Figure 2—figure supplement 1). However, neither SAM nor SAH were observed to be bound to METTL14 in the METTL3-14 complex structures. Modeling a molecule of SAM in the METTL14 active site reveals that the binding would be precluded by steric clashes between the adenine moiety and the side chains of and in the absence of structural in METTL14. METTL14 lacks residues that could form contacts to the ribose in SAM, as residues Asn549METTL3 and to and respectively. Additionally, the putative acceptor binding site in METTL14 is by the side chains of and from loops to ASL1 and ASL2 in METTL3 and respectively) (Figure The equivalent of the METTL3 DPPW motif, is thus not and lacks an aromatic moiety required for the stacking interaction with the acceptor adenine, (Figure these that METTL14 contains a degenerate active site that is unable to donor and acceptor that METTL14 is catalytically To this we putative active site residues implicated in donor substrate binding and in METTL3 and METTL14 by and the methyltransferase activities of complexes using an RNA oligonucleotide containing the consensus m6A methylation sequence of or to alanine reduced the methyltransferase activity of the complex to background (Figure In alanine of or the putative active site in METTL14 had little effect on the MTase activity of the METTL3-14 complex (Figure These results indicate that the catalytic activity of the complex is to the METTL3 active site and that METTL14 likely plays a non-catalytic role within the m6A writer complex. Figure Download asset Open asset METTL14 contains a degenerate active site and is catalytically (A) view of the putative methyltransferase active site in METTL14. The binding of the SAM donor in was by superimposing the structure of METTL3. (B) Methyltransferase activities of the METTL3-METTL14 complex and active site alanine RNA binding by the METTL3-METTL14 dimer the structure of the METTL3-14 complex with structures of EcoP15I and bound to DNA substrates et al., et al., 2015) suggests two binding for the RNA acceptor on the acceptor adenine is superimposed onto the active site in METTL3 or the site in METTL14 (Figure the acceptor adenine bound in the active site of METTL3, the of the RNA substrate is over a located at the interface of the METTL3 and METTL14 subunits (Figure is highly conserved and residues and and The four residues form a pocket by a co-crystallized RNA group binding (Figure These that this surface substrate RNA binding and are in good agreement with the presence of bound donor substrate and product in the METTL3 active site and of catalytic activity in active site of METTL3. In the surface to the putative active site in METTL14 on the opposite of the METTL3-14 dimer lacks evolutionary and is (Figure substrate RNA binding by this surface and further the that METTL14 is catalytically To the that the to the METTL3 active site at the METT3-METTL14 dimer interface is involved in substrate RNA we with alanine and the complex for MTase The activity of the complex was substantially than in the (Figure was additionally to the catalytic activity of complex was reduced to background further suggesting that these residues are involved in substrate RNA binding (Figure We additionally the of the amino acid residues in the putative acceptor binding in METTL3 and METTL14. substitution of Tyr406METTL3 reduced the MTase activity of the complex to background In substitution of had little effect on MTase activity (Figure these results that the m6A writer complex contains only one active site in METTL3 and that substrate RNA binding is mediated by a conserved surface at the interface of the METTL3-14 subunits. Figure Download asset Open asset The METTL3-METTL14 dimer interface substrate (A) Model of acceptor substrate binding in the active site of METTL3 by superimposing METTL3 in the METTL3-METTL14 complex structure with the active methyltransferase subunit in the structure of the of the N6-adenosine DNA methyltransferase EcoP15I bound to substrate only the acceptor of the substrate DNA is METTL3-METTL14 is shown in surface with subunits colored as in Figure (B) Model of acceptor substrate binding to METTL14 by superimposing METTL14 in the METTL3-METTL14 complex structure with the active methyltransferase subunit in the substrate complex. (C) and sequence shown for the side of the METTL3-METTL14 heterodimer containing the METTL3 active site. The and SAM co-crystallized with the as well as the acceptor on EcoP15I structure are shown in yellow. The surface was in sequence was using the et al., (D) surface and sequence for the side of the complex to the degenerate METTL14 catalytic site. (E) view of the binding to interact with the of substrate Methyltransferase activities of WT METTL3-METTL14 complex and the alanine of putative RNA substrate binding The activities were measured as in Figure Methyltransferase activities of WT and complex. Discussion recent studies have that m6A plays roles in eukaryotic RNA metabolism that impact aspects of cellular Despite in understanding the biological of have been only limited mechanistic insights into the molecular that this epitranscriptomic In this we have the catalytic core of the m6A writer complex and determined the structure of its complexes with methyl group donor substrates and findings are in good agreement with other recent structural studies of the m6A writer complex (Wang et al., 2016a, 2016b). The molecular architecture of the METTL3-METTL14 dimer is similar to that of homodimeric bacterial N6-adenosine DNA methyltransferases such as at an evolutionary in which the m6A writer emerged by the of a bacterial homodimeric DNA and sequence of the METTL3 and METTL14 subunits have the specificity The presence of an