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Structures and mechanisms of tRNA methylation by METTL1–WDR4

Nature volume 613, pages 383–390 (2023)Cite this article

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Abstract

Specific, regulated modification of RNAs is important for proper gene expression1,2. tRNAs are rich with various chemical modifications that affect their stability and function3,4. 7-Methylguanosine (m7G) at tRNA position 46 is a conserved modification that modulates steady-state tRNA levels to affect cell growth5,6. The METTL1–WDR4 complex generates m7G46 in humans, and dysregulation of METTL1–WDR4 has been linked to brain malformation and multiple cancers7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22. Here we show how METTL1 and WDR4 cooperate to recognize RNA substrates and catalyse methylation. A crystal structure of METTL1–WDR4 and cryo-electron microscopy structures of METTL1–WDR4–tRNA show that the composite protein surface recognizes the tRNA elbow through shape complementarity. The cryo-electron microscopy structures of METTL1–WDR4–tRNA with S-adenosylmethionine or S-adenosylhomocysteine along with METTL1 crystal structures provide additional insights into the catalytic mechanism by revealing the active site in multiple states. The METTL1 N terminus couples cofactor binding with conformational changes in the tRNA, the catalytic loop and the WDR4 C terminus, acting as the switch to activate m7G methylation. Thus, our structural models explain how post-translational modifications of the METTL1 N terminus can regulate methylation. Together, our work elucidates the core and regulatory mechanisms underlying m7G modification by METTL1, providing the framework to understand its contribution to biology and disease.

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Fig. 1: Architecture of the human METTL1–WDR4 complex.
Fig. 2: Architecture of the METTL1–WDR4–tRNA complex in three states.
Fig. 3: Structural recognition of tRNA shape by METTL1–WDR4.
Fig. 4: Detailed view of the active site.
Fig. 5: METTL1 N terminus coordinates SAH binding with RNA and protein conformational changes.
Fig. 6: Mechanistic model for tRNA m7G46 methylation by METTL1–WDR4.

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Data availability

The atomic models for METTL1–WDR4 (8D58), METTL1–WDR4–tRNA (8D9K), METTL1–SAM (8D59), METTL1–SAH (8D5B), METTL1–WDR4–tRNA–SAM (8D9L) and METTL1–WDR4–tRNA–SAH (8EG0) are deposited in the PDB (https://www.rcsb.org/). Cryo-EM maps and masks of METTL1–WDR4–tRNA (EMD-27264), METTL1–WDR4–tRNA–SAM (EMD-27265) and METTL1–WDR4–tRNA–SAH (EMD-28108) used to build the models are deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/).

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Acknowledgements

We thank the Cryo-Electron Microscopy Facility (Cancer Prevention Research Institute of Texas (CPRIT) RP170644) and the Structural Biology Laboratory (CPRIT RP220582) at UT Southwestern for support with synchrotron and cryo-EM data collection. The use of the SBC 19-ID beamline at Advanced Photon Source is supported by the US Department of Energy contract DE-AC02-06CH11357. This work was supported by the US National Institutes of Health (R01GM122960 and R01CA258589), CPRIT (RP190259) and the Welch Foundation (I-2115-20220331). Y.N. is a Packard Fellow, Pew Scholar and Southwestern Medical Foundation Scholar in Biomedical Research.

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Authors and Affiliations

  1. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Victor M. Ruiz-Arroyo, Rishi Raj, Kesavan Babu, Otgonbileg Onolbaatar, Paul H. Roberts & Yunsun Nam

  2. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Victor M. Ruiz-Arroyo, Rishi Raj, Kesavan Babu, Otgonbileg Onolbaatar, Paul H. Roberts & Yunsun Nam

  3. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Victor M. Ruiz-Arroyo, Rishi Raj, Kesavan Babu, Otgonbileg Onolbaatar, Paul H. Roberts & Yunsun Nam

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Contributions

Y.N. conceived and supervised the study. V.M.R.-A. prepared the cryo-EM grids, collected data, determined cryo-EM structures and refined the atomic models using cryo-EM maps. R.R. prepared crystals and collected diffraction data. R.R. and K.B. performed data processing, model building and refinement for the crystal structures. V.M.R.-A., R.R., K.B., O.O. and P.H.R. produced recombinant proteins and RNAs and performed biochemical assays. V.M.R.-A. and Y.N. prepared the manuscript with help from all other co-authors.

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Correspondence to Yunsun Nam.

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Extended data figures and tables

Extended Data Fig. 1 METTL1-WDR4 protein purification and tRNA complex reconstitution.

a, SDS-PAGE of purified full-length wild-type METTL1-WDR4 complex. A representative gel among 3 replicates is shown. b, EMSA to measure the affinity for tRNALys. For each gel, protein concentrations are 0, 16, 32, 65, and 130 nM, left to right. Representative gel among 3 replicates is shown. c, Quantification of EMSA for METTL1-WDR4 from 3 replicate experiments. d, Superimposition of yeast Trm8-Trm82 (PDB 2VDU, orange) onto the crystal structure of human METTL1-WDR4. The complex structures were superimposed by aligning METTL1 with Trm8. e-g, Superimposition of METTL1 structures as indicated. The structures missing PDB codes are from this study.

Extended Data Fig. 2 Cryo-EM data processing for the METTL1-WDR4-tRNALys structure.

a, Cryo-EM data processing workflow. Left, a representative micrograph of the METTL1-WDR4-tRNA complex particles. A total of 16,993 images were used for picking and 2D classification. 2D class averages that showed high-resolution features were used for further 3D analysis using Cryosparc. 3D classification identified different populations that showed partial or no density around the anticodon arm. 3D classification applying the indicated mask revealed a homogeneous population of particles showing contiguous density in the tRNA region. b, Angular distribution plot. c, Local resolution map shown with colors on the sharpened map. d, Directional FSC plot and FSC curves showing the resolution at 0.143 cutoff.

Extended Data Fig. 3 Cryo-EM data processing for the METTL1-WDR4-tRNALys-SAM structure.

a, Cryo-EM data processing workflow. Left, a representative micrograph of the METTL1-WDR4-tRNA-SAM complex particles. A total of 5,607 images were used for picking and 2D classification. 2D class averages that showed high-resolution features were used for further 3D analysis using Cryosparc. 3D classification identified different populations that showed partial density around the anticodon arm. 3D classification applying the indicated mask revealed a homogeneous population of particles showing contiguous density in the tRNA region. b, Angular distribution plot. c, Local resolution map shown with colors on the sharpened map. d, Directional FSC plot and FSC curves showing the resolution at 0.143 cutoff.

Extended Data Fig. 4 Cryo-EM data processing for the METTL1-WDR4-tRNALys-SAH structure.

a, Cryo-EM data processing workflow. Left, a representative micrograph of the METTL1-WDR4-tRNA-SAH complex particles. A total of 6,120 images were used for picking and 2D classification. 2D class averages that showed high-resolution features were used for further 3D analysis using Cryosparc. 3D variability analysis identified a population of particles that showed consistent density for the WDR4 C-terminal helix and several rounds of 3D Heterogeneous Refinement identified particles showing prominent density for G46. b, Angular distribution plot. c, Local resolution map shown with colors on the sharpened map. d, Directional FSC plot and FSC curves showing the resolution at 0.143 cutoff.

Extended Data Fig. 5 Conformational changes in structures containing the METTL1-WDR4-tRNA complex.

a, Superimposition of the METTL1-WDR4 crystal structure on the METTL1-WDR4-tRNA cryo-EM structure, aligned by METTL1. b, Flexible loop of METTL1 (161–175, catalytic loop) becomes more ordered with RNA. c, Superimposition of different tRNALys structures. All three have the same sequence except the tips of the anticodon arm and the acceptor arm. The B. Taurus structure (PDB:1FIR) was with a fully modified tRNA and the other two structures were obtained for unmodified RNA after in vitro transcription. d, Superimposition of the METTL1-WDR4 crystal structure (gray) onto the SAH-bound quaternary complex cryo-EM structure (multiple colors), aligned by WDR4. Movement of the WDR4 C-terminal helix upon binding RNA is shown with a dashed arrow. e, Superimposition of all three cryo-EM structures (colored by state) presented in this study, aligned by WDR4. f. Surface representation of the SAH-bound cryo-EM structure colored by evolutionary sequence conservation (Consurf server). The orientation is identical to Fig. 3a and b.

Extended Data Fig. 6 Structural and sequence organization of tRNA.

a-b, Sharpened cryo-EM map (mesh) near the SAH-binding site (a) and the G46 binding pocket (b). c, Sequence alignment of the human tRNAs used in this study shaded by conservation. Red boxes indicate nucleobases within 4 Å of protein. Sequences were aligned using Clustal Omega and visualized by Geneious Prime. d, In vitro methylation activity of full-length METTL1-WDR4 for the indicated tRNAs with the specified variable loop sequences, shown as mean ± SD from 3 replicates. e, EMSA using METTL1-WDR4 with different tRNAs shows no dramatic differences in affinities. Representative images from 3 replicate experiments are shown. For each gel, protein concentrations are 0, 16, 32, 65, and 130 nM, left to right.

Extended Data Fig. 7 Sequence alignment of METTL1 protein homologs.

Sequences used are from human (Q9UBP6), Mus musculus (Q9Z120), Bos taurus (Q2YDF1), Xenopus laevis (Q6NU94), Danio rerio (Q5XJ57), Drosophila melanogaster (O77263), Caenorhabditis elegans (Q23126) and Saccharomyces cerevisiae (Q12009) METTL1. Sequences were aligned using Clustal Omega and visualized by Geneious Prime. Residues within 4 Å of RNA in different states are indicated with asterisks.

Extended Data Fig. 8 Sequence alignment of WDR4 protein homologs.

Sequences used are from human (P57081), Mus musculus (Q9EP82), Bos taurus (A7E3S5), Xenopus laevis (Q7ZY78), Danio rerio (A4IGH4), Drosophila melanogaster (Q9W415), Caenorhabditis elegans (Q23232) and Saccharomyces cerevisiae (A6ZYC3) WDR4. Sequences were aligned using Clustal Omega and visualized by Geneious Prime. Residues within 4 Å of RNA in different states are indicated with asterisks.

Extended Data Table 1 Crystallography Data Collection and Refinement Statistics
Full size table
Extended Data Table 2 Cryo-EM Data Collection, Refinement, and Validation Statistics
Full size table

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Ruiz-Arroyo, V.M., Raj, R., Babu, K. et al. Structures and mechanisms of tRNA methylation by METTL1–WDR4. Nature 613, 383–390 (2023). https://doi.org/10.1038/s41586-022-05565-5

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