Abstract
Chemical modifications of RNA have key roles in many biological processes1,2,3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4,5,6,7. The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8,9,10,11,12,13,14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15,16,17. How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show, through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity.
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Data availability
The cryo-EM structures of METTL1–WDR4–tRNAPhe and METTL1–WDR4–tRNAVal, and X-ray crystal structure of METTL1–WDR4 have been deposited to the PDB under accession numbers 8CTH, 8CTI and 7U20, respectively. The cryo-EM density maps of METTL1–WDR4–tRNAPhe and METTL1–WDR4–tRNAVal have been deposited in the Electron Microscopy Data Bank under accession numbers 26990 and 26991. Several structural coordinates in the PDB database were used in this study, which can be located through accession numbers 1EHZ, 2VDU, 3JAG, 3CKK, 7OGJ, 2VDV and 7PL1. NMR resonance assignments were deposited to the Biological Magnetic Resonance Data Bank under accession number 51362.
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Acknowledgements
E.A.O. is supported by the Pew Latin American Fellows Program in the Biomedical Sciences from Pew Charitable Trusts and by a fellowship from the Damon Runyon Cancer Research Foundation (DRG-2378–19). R.I.G. is supported by an Outstanding Investigator Award (R35CA232115) from the National Cancer Institute of the US National Institutes of Health. This work was in part supported by National Institutes of Health grants R01 CA218278 and R01 CA214608 (to E.S.F.), the Mark Foundation for Cancer Research (Mark Foundation Emerging Leader Award 19-001-ELA to E.S.F.) and the Cancer Research Institute (Irvington Postdoctoral Fellowship CRI 3442 to S.S.R.B.). This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. We thank the staff at the Harvard Cryo-EM Center for Structural Biology for their support during grid screening and data collection. This research used resources of the Advanced Photon Source, a US Department of Energy Office of Science User Facility operated for the Department of Energy Office of Science by Argonne National Laboratory under contract number SE-AC02-06CH11357. We acknowledge the SBGrid consortium for assistance with high-performance computing.
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Contributions
J.L. and R.I.G. conceived the project. J.L. performed recombinant protein purification and ternary complex reconstitution. J.L., L.W. and P.F. prepared and optimized samples for cryo-EM. L.W. performed cryo-EM data processing. J.L. built models, with assistance from L.W., S.S.R.B. and M.H. J.L., R.P.N. and E.S.F. determined and refined the crystal structure of the binary complex. J.L., Q.H., T.V. and H.Y. performed biochemical experiments. E.A.O., J.L. and Q.H. performed cellular experiments. R.I.G., E.S.F., H.A. and H.W. supervised the project. All authors organized and analysed the data. J.L., L.W. and R.I.G. wrote the manuscript with input from all authors.
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R.I.G. is a co-founder and scientific advisory board (SAB) member of 28/7 Therapeutics and Theonys. E.S.F. is a founder, SAB member and equity holder in Civetta Therapeutics, Lighthorse Therapeutics, Neomorph, Inc. (board of directors) and Proximity Therapeutics. E.S.F. is a SAB member and equity holder in Avilar Therapeutics and Photys Therapeutics. E.S.F. is also a consultant to Novartis, Sanofi, EcoR1 capital, Avilar and Deerfield. The Fischer laboratory receives or has received research funding from Astellas, Novartis, Voronoi, Interline, Ajax and Deerfield. The rest of the authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Sample preparation and quality check.
(a–b) tRNA candidates for ternary complex reconstitution. Schematic representations of yeast tRNAPhe and human tRNAVal. Yeast tRNAPhe is a matured tRNAPhe purified from yeast (Sigma) (a). Human tRNAVal is an annealed single strand RNA oligos synthesized based on human tRNAVal-TAC sequence (Horizon) (b). (c) Chromatography traces and SDS-PAGE analysis of purified METTL1-WDR4 complex (absorption at 280nm and 254nm). For gel source data, see Supplementary Fig. 3. (d) Gel filtration profiles of free tRNAPhe (blue), METTL1-WDR4 binary complex (green), METTL1-WDR4-tRNAPhe ternary complex (purple) and METTL1-WDR4-tRNAVal ternary complex (yellow) (absorption at 280nm are shown). (e) Identification of the reconstituted METTL1-WDR4-tRNAPhe ternary complex. Sample in the main peak of the reconstitution chromatography trace is analyzed by native PAGE. tRNA and protein are virtualized by EB and G250 staining separately. For gel source data, see Supplementary Fig. 4.
Extended Data Fig. 2 Cryo-EM workflows and the quality of reconstructed cryo-EM maps.
(a–b) Workflow of 3D reconstruction of METTL1-WDR4-tRNAPhe dataset (a) and the METTL1-WDR4-tRNAVal dataset (b). (c–d) Fourier shell correlation (FSC) curves (upper panel) and orientation distributions (lower panel) of 3D reconstructed METTL1-WDR4-tRNAPheand METTL1-WDR4-tRNAVal cryo-EM maps. See also Extended Data Table 1. (e) Histogram of directional FSC curves of METTL1-WDR4-tRNAPhe dataset (upper panel) and the METTL1-WDR4-tRNAVal dataset (lower panel). (f) Local resolutions of METTL1-WDR4-tRNAPhe (upper panel) and METTL1-WDR4-tRNAVal (lower panel) cryo-EM maps. (g) Representative segments of sharpened cryo-EM map fitted with the model. (h) State A and State B models of METTL1-WDR4-tRNAPhe fit in sharpened cryo-EM maps. The variable loop of tRNA fitted with sharpened (top) and unsharpened (bottom) maps from 3D variability analysis are shown in parallel with the whole model.
Extended Data Fig. 3 Similar binding mode of METTL1-WDR4 to tRNAPhe and tRNAVal.
(a) Cryo-EM density maps of METTL1-WDR4-tRNAVal complex and the corresponding atomic model. The unsharpened (left), sharpened (middle) and DeepEMhancer processed (right) density maps are shown. (b) Overall structural superposition of METTL1-WDR4-tRNAVal (slate) and METTL1-WDR4-tRNAPhe (white). (c) Electrostatic potential of METTL1 and WDR4 in ternary complex (tRNAPhe). Red, negative; blue, positive. Figure was generated using PyMOL. tRNA domains are colored according to Extended Data Fig. 1a. (d) Tilted loading of tRNAPhe onto the METTL1-WDR4 complex. Overall structure of METTL1-WDR4-tRNAPhe complex from the WDR4 side. The angle between the short axis of METTL1-WDR4 and the aminoacyl-branch axis of tRNA is about 130 degrees. The angle is measured utilizing residue 213 (WDR4), residue 163 (WDR4) and base 73 of tRNA using PyMOL.
Extended Data Fig. 4 Sequence alignment of METTL1 proteins.
The human METTL1 protein sequence was aligned with its respective homologs. The secondary structure diagram for human METTL1 is shown on the top. Conserved residues are shaded in yellow, whereas essentially invariant residues are shown in red. The conserved N-terminal region, aC and a6 helix are underlined. K143 (key residue that interacts with WDR4) is highlighted with a blue star on the bottom. The alignment is performed with the Clustal Omega multiple sequence alignment program (EMBL-EBI) and visualized by ESPript 3.0 server.
Extended Data Fig. 5 Domain organization and sequence alignment of WDR4 proteins.
(a) Schematic representation of full-length WDR4 domains based on sequence, secondary structure prediction and experimental structures. WD, WD family repeat domain and are numbered with B1–B7. (b) Sequence alignment of WDR4 proteins. The secondary structure diagram (black, based on experimental structure; pale green, based on AlphaFold prediction) for human WDR4 is shown on the top. Conserved residues are shaded in yellow, whereas essentially invariant residues are shown in red. B3 and B4 are underlined in green and blue, respectively. Key residues are highlighted with stars on the bottom. Orange stars, residues involved in tRNA (T-arm) binding; magenta stars, patient related mutagenesis sites; blue stars, METTL1 interaction sites. The alignment is performed with the Clustal Omega multiple sequence alignment program (EMBL-EBI) and visualized by ESPript 3.0 server. (c) Structure of METTL1-WDR4-tRNAPhe ternary complex with top view (only METTL1 and WDR4 are shown). The conserved region of B2–B5 (WDR4) is highlighted in red.
Extended Data Fig. 6 The conformational change of METTL1-WDR4 upon tRNA binding.
(a)Structure comparison between METTL1-WDR4-tRNAPhe and METTL1-WDR4. The structures are superposed on WDR4 protein. The tRNA in the ternary complex is not shown for a better view. METTL1-WDR4-tRNAPhe, pink; METTL1-WDR4, Cyan. The α1, α2, α5 and α6 helices shift toward WDR4 and tRNA side. Structural changes of the residues 164–173 fragment of METTL1 are highlighted with a dash line box. (b) Superposition of tRNA-free (cyan) and tRNA bound (pink) states of METTL1. The structures are superposed on METTL1 protein and only only METTL1 are shown. The loop (residues 164–173) connecting α1 and the core fold of METTL1 forms the αC helix upon tRNA binding. (c) Binary complex model from METTL1-WDR4-tRNAPhe cryo-EM dataset fit in sharpened (top) and unsharpened (bottom) cryo-EM maps. (d) Structure comparison between EM_Binary (teal) and crystal METTL1-WDR4 (gray). The METTL1 protein is superimposed. (e) Local resolution of binary complex map. The αC loop of METTL1 region is highlighted with dashed line circle. (f)The αC loop of METTL1 (EM_Binary) fit in sharpened (top) and unsharpened (bottom) cryo-EM maps.
Extended Data Fig. 7 Essential residues of METTL1-WDR4 for MTase activity and tRNA recognition.
(a) Magnified view of METTL1-WDR4 interface in the crystal binary complex structure. The interactions of key residues are shown in dashed lines. K143 (METTL1) forms a salt bridge with D166 (WDR4); hydrogen bonds are formed between Y37 (METTL1) and E167 (WDR4), N147 (METTL1), and K168 (WDR4), K40 (METTL1) and mainchain of L185 (WDR4). (b) Relative methyltransferase activity of METTL1-WDR4 complexes expressed with indicated mutations. WT, wild type; Mut, catalytic dead double mutant (L160A;D163A). Two technical replicates were performed. (c–d) In vivo rescue experiment with METTL1 or WDR4 carrying indicated mutations in Wdr4 (KO) cell lines (c) or Mettl1 (KO) cell lines (d). n = 2, biologically independent samples. Expression of WT and variants METTL1 or WDR4 is checked by western blot (lower panel). For gel source data, see Supplementary Fig. 5-6. (e) Structure comparison between METTL1-WDR4 crystal structure (PDB 7U20, METTL1, splitpea; WDR4, light blue) and Trm8-Trm82 (PDB 2VDU, orange). The WDR4 protein is superimposed. (f) Distance measurement between METTL1 E183 (OE2) and WDR4 R170 (NH1) in the crystal binary complex structure. 2Fo-Fc map is shown (1.2 σ). Figures were generated in Coot. (g, h) Western blot detection of overexpressed METTL1 or WDR4 in rescue experiments relative to Fig 2e, j. n = 2, biologically independent samples. For gel source data, see Supplementary Fig. 7.
Extended Data Fig. 8 The METTL1 N-terminus plays important roles in catalytic regulation.
(a) Superimposed METTL1 N-terminus of available structures. The first visible residues of human METTL1 are labeled. (b) The IDDT value of the predicted METTL1-WDR4 structure. (c) The interactions between the N-terminal and α2 helix in AlphaFold prediction. (d) AlphaFold predicted METTL1 is superposed onto METTL1-WDR4-tRNAPhe. The predicted residues 16–21 insert into the space between METTL1 and tRNA. (e) Residue-specific secondary structure propensities derived from 1HN, 15N, 13C’, 13Cα and 13Cβ chemical shifts assignments. α-helix (red), coil/unstructured (grey), β-strand (green). (f) Competitive TR-FRET binding assay of labeled full-length METTL1-WDR4 with unlabeled proteins. The determined IC50 is listed. Two technical replicates were performed. (g) Western blot detection of overexpressed METTL1 or WDR4 relative to Fig. 3f. n = 2, biologically independent samples. For gel source data, see Supplementary Fig. 7. (h) In vivo rescue experiment with indicated mutations in Mettl11 (KO) cell lines (left). Expression of protein is checked by western blot (right). For gel source data, see Supplementary Fig. 8. (i) Key components of the G46 binding cavity fit in sharpened METTL1-WDR4-tRNAPhe cryo-EM map (mesh). Key residues and cofactor SAH are shown in stick. (j) Docking model of G46 flipping into the catalytic pocket with SAM bound. The transferred methyl group is indicated by arrow. Relevant elements are adjusted manually to make 180° angle and 2 Å between the guanine-N7 and S-CH3. The potential interactions between METTL1 and the base of G46 are highlighted with dashed lines. (k) Schematic diagram of the docking model depicting potential interactions (dashed lines) between G46 and SAM in the METTL1 active site prior to methyl transfer. (l-m) Relative methyltransferase activity of METTL1-WDR4 with buffer pH ranging from 5.6 to 8.33 (l) and indicated mutation (m). Two technical replicates were performed.
Supplementary information
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This file contains Supplementary Figures 1–8.
Supplementary Video 1
Three-dimensional variability analysis of METTL1-WDR4-tRNAPhe complex. 3D variability analysis was generated in cryoSPARC (filter resolution 5 Å and number of modes 3) based on the non-uniform refinement indicated in Extended Data Fig. 2a. This analysis showed that the anticodon-arm of tRNAPhe was bent towards METTL1 and accompanied with local unwinding of the variable loop, while the rest of the tRNAPhe remained unchanged relative to METTL1-WDR4 complex.
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Li, J., Wang, L., Hahn, Q. et al. Structural basis of regulated m7G tRNA modification by METTL1–WDR4. Nature 613, 391–397 (2023). https://doi.org/10.1038/s41586-022-05566-4
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DOI: https://doi.org/10.1038/s41586-022-05566-4
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