Abstract

N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1,2,3,4,5. m6A modification is catalysed by METTL3 and enriched in the 3′ untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5′ cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs—including bromodomain-containing protein 4—that is also m6A-modified in human primary lung tumours. The METTL3–eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3–eIF3h as a potential therapeutic target for patients with cancer.

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Data availability

The m6A meRIP–seq and RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE117299. All other data are available from the authors on request.

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Acknowledgements

Thanks to Y. K. Kim for CBP80 and CTIF antibodies. S.L. was supported by a Damon Runyon-Sohn Pediatric Fellowship (DRSG-7–13) and a grant from Alex’s Lemonade Stand Foundation. R.I.G. was supported by grants from the US National Institute of General Medical Sciences (NIGMS) (R01GM086386) and National Cancer Institute (NCI) (R01CA211328).

Reviewer information

Nature thanks C. Mason and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • Wantae Kim

    Present address: Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea

  1. These authors contributed equally: Junho Choe, Shuibin Lin

Affiliations

  1. Stem Cell Program, Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA

    • Junho Choe
    • , Shuibin Lin
    • , Qi Liu
    • , Julia Ramirez-Moya
    • , Peng Du
    •  & Richard I. Gregory
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    • Junho Choe
    • , Shuibin Lin
    • , Qi Liu
    • , Longfei Wang
    • , Peng Du
    • , Piotr Sliz
    •  & Richard I. Gregory
  3. Center for Translational Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

    • Shuibin Lin
  4. Department of Pathology, Cancer Center, Beth Israel Deaconess Medical Center, Boston, MA, USA

    • Wencai Zhang
    •  & Frank J. Slack
  5. Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain

    • Julia Ramirez-Moya
    •  & Pilar Santisteban
  6. Harvard School of Dental Medicine, Boston, MA, USA

    • Wantae Kim
  7. Innovation Center for Biomedical Informatics, Georgetown University Medical Center, Washington, DC, USA

    • Shaojun Tang
  8. Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA

    • Shaojun Tang
  9. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Rani E. George
  10. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    • Rani E. George
    •  & Richard I. Gregory
  11. Department of Surgery, Brigham and Women’s Hospital, Boston, MA, USA

    • William G. Richards
  12. Division of Hematology and Medical Oncology, NYU School of Medicine, New York, NY, USA

    • Kwok-Kin Wong
  13. School of Biosciences and Medicine, University of Surrey, Guildford, UK

    • Nicolas Locker
  14. Harvard Initiative for RNA Medicine, Boston, MA, USA

    • Frank J. Slack
    •  & Richard I. Gregory
  15. Harvard Stem Cell Institute, Cambridge, MA, USA

    • Frank J. Slack
    •  & Richard I. Gregory

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Contributions

J.C., S.L. and R.I.G. designed the research; J.C., S.L., W.Z., L.W., J.R.-M. and W.K. performed all the experiments; J.C. performed in vitro translation assay, tethering assay, polysome fractionation and RNA-seq, co-immunoprecipitation, electron microscopy, cap-association assay and in situ PLA; S.L. performed GST pull-down assay, far-western blotting, immunohistochemical staining, cell invasion assay and m6A meRIP–seq; W.Z. performed soft agar colony formation assays and in vivo tumour xenograft; L.W. performed electron microscopy; J.R.-M. performed cell proliferation and apoptosis assays; W.K. performed in situ PLA; S.L., Q.L., P.D. and S.T. performed all bioinformatics analysis; W.G.R. and K.-K.W. provided human lung cancer patient samples; N.L. provided eIF3 complex. P.Sl., P.Sa., R.E.G. and F.J.S. contributed to discussion. J.C., S.L. and R.I.G. analysed data and wrote the paper with input from other authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Richard I. Gregory.

Extended data figures and tables

  1. Extended Data Fig. 1 METTL3 binding close to the stop codon enhances translation.

    a, Schematic of reporter plasmids containing Firefly luciferase cDNA and different positions of MS2 binding sites. b, Western blotting with indicated antibodies. Blot is representative of two independently performed experiments with similar results. c, RT–qPCR analysis of reporter mRNAs. Each tested reporter mRNAs were normalized to RLuc mRNAs. The FLuc:RLuc ratio for each construct with Flag–MS2 expression was set to 1. Data are mean ± s.d. from three biologically independent samples. d, Tethering assay to measure translation efficiency as described in Fig. 1h. Data are mean ± s.d. from three biologically independent samples. P values from a two-sided t-test. e, Colloidal Coomassie blue staining of recombinant protein His–Flag–MS2, His–Flag–MS2–METTL3, or His–Flag–MS2–METTL3 (1–200). f, Ethidium bromide-stained agarose gel electrophoresis of the indicated in vitro transcribed reporter mRNAs; FLuc-MS2bs without poly (A) tail (Poly (A) -) or FLuc-MS2bs with 30-nt poly (A) tail (Poly (A) +). f, g, Images are representative of two independently performed experiments with similar results. g, In vitro translation of reporter mRNAs using either H1299 cell extracts or rabbit reticulocyte lysate (RRL). The levels of in vitro-translated FLuc protein were analysed using luciferase assays. Value of FLuc activity in the presence of His–Flag–MS2 recombinant protein was set to 1. Data are mean ± s.d. from six independent experiments. P value is from a two-sided t-test; ***P < 0.001, multiple comparison.

  2. Extended Data Fig. 2 N-terminal region of METTL3 promotes translation.

    a, Schematic of METTL3-deletion mutants or mutation in METTL3 catalytic domain. b, Western blotting with indicated antibodies. The blot is representative of two independently performed experiments with similar results. c, RT–qPCR analysis of reporter mRNAs. FLuc–MS2bs mRNA levels were normalized to RLuc mRNAs. The FLuc:RLuc ratio obtained in Flag–MS2 (control) was set to 1. Data are mean ± s.d. from three biologically independent samples. d, Tethering assay to measure translation efficiency of reporter mRNAs as described in Fig. 1h. Data are mean ± s.d. from three biologically independent samples. P value is from a two-sided t-test; **P < 0.01, multiple comparison.

  3. Extended Data Fig. 3 METTL3 associates with translation initiation factors.

    a, Deletion mutants of METTL3 were expressed in HeLa cell. The total-cell extracts (Input) and the cap-associated protein samples were analysed by western blotting using the indicated antibodies. b, Cap-association assay with METTL3 depletion. The total-cell extracts (Input) and the cap-bound protein samples were analysed by western blotting using the indicated antibodies. m7GpppG cap analogue was used for antagonizing cap-associating proteins binding to m7GTP-agarose. c, Same as b, except HeLa cells were transfected with CTIF, EIF3B or EIF4G1 siRNA. In a, b, blots are representative of two independently performed experiments with similar results. d–f, Mass spectrometry of Flag–METTL3 interacting proteins. d, Proteins that were co-immunopurified with Flag–METTL3 subjected to 4–12% Tris–glycine SDS–PAGE. Colloidal Coomassie blue staining was performed. e, Gene Ontology analysis of the identified proteins from mass spectrometry. One experiment was performed. Hypergeometric distribution (one-tail) with Bonferroni adjustment was used to determine enrichment statistical significance. f, Table showing the translation involving factors identified from mass spectrometry.

  4. Extended Data Fig. 4 N-terminal region of METTL3 directly interacts with MPN domain of eIF3h.

    a, Electron-microscopy images of polyribosome with gold-particle labelling of METTL3. Red arrows indicate METTL3 with immuno-gold particle (6 nm). Images are representative of three independent experiments with similar results. b, Counting of METTL3 with gold-particle labelling in each polyribosome. c, Electron-microscopy images of polyribosome with METTL3 and eIF4E. Red arrows indicate METTL3 with immuno-gold particle (6 nm) and yellow arrows indicate eIF4E with immuno-gold particle (10 nm). Images are representative of four independently performed experiments with similar results. d, Mean distance between immuno-gold particles was measured. Data are mean ± s.d. of six biologically independent samples from at least three independent experiments. e, Colloidal Coomassie blue staining of recombinant protein His–METTL3 or His–METTL3 1–200 amino acid fragments. f, Colloidal Coomassie blue staining of recombinant GST-tagged protein eIF3g, eIF3h, eIF3i, eIF3j or eIF3m. g, GST–eIF3h was co-purified with His–METTL3 in the presence of either rabbit IgG (rIgG) or anti-METTL3 antibody. Levels of co-purified His–METTL3 were analysed by western blotting. eg, Images are representative of two independently performed experiments with similar results. h, Schematic diagram of human eIF3h deletion mutants. i, Colloidal Coomassie blue staining of recombinant GST–eIF3h, GST–eIF3h (1–222) or GST–eIF3h (29–222). j, GST pull-down of the indicated eIF3h deletion mutants. Co-purified His–METTL3 was analysed by western blotting. i, j, One experiment was performed. k, Western blotting demonstrates efficient knockdown of eIF3h protein. Blots are representative of three independently performed experiments with similar results. l, RT–qPCR analysis demonstrates efficient down regulation of eIF3h mRNA. Data are mean ± s.d. from three biologically independent samples. P values are from a two-sided t-test. m, RT–qPCR analysis of reporter mRNAs. FLuc–MS2bs reporter mRNAs were normalized to RLuc mRNAs. The FLuc:RLuc ratio obtained in Flag–MS2 was set to 1. Data are mean ± s.d. from three biologically independent samples.

  5. Extended Data Fig. 5 METTL3 has no significant effect on mRNA stability.

    a, Western blotting with indicated antibodies. Blot is representative of three independently performed experiments with similar results. b, Gene Ontology analysis of the overlapping mRNAs (n = 809) in Fig. 2d. Hypergeometric distribution (one-tail) with Bonferroni adjustment was used to determine statistical significance of enrichment. c, RT–qPCR analysis using indicated primers. Data are mean ± s.d. from three technical replicates. d, e, Half-life of endogenous mRNAs was analysed by RT–qPCR using indicated primers. Data are mean ± s.e.m. from six independent experiments. d, P values are from a two-sided t-test; multiple comparison for the P values showed that there were no significant differences between the samples for all the tested mRNAs (P > 0.05).

  6. Extended Data Fig. 6 Widespread role of METTL3 in oncogene translation.

    a, Immunoprecipitation of endogenous METTL3 and western blotting using the indicated antibodies. The blot is representative of two independently performed experiments with similar results. b, Density plot reflects the distribution of changes in percentage spliced in ∆PSI values and corresponding P values for alternative splicing events detected by rMATs v.3.2.5 (rMATs is developed on the basis of a hierarchical framework and likelihood-ratio test was used to detect differential splicing). Splicing events at a FDR < 5% and ΔPSI > 0.1 are considered as significant. Black dots indicate total mRNAs. Red dots (4,276 mRNAs) indicate mRNAs in METTL3-depleted cells that are translated more than twofold less. c, Western blot using indicated antibodies in control-, METTL3- or YTHDF1-knockdown cells. Blots are representative of two independently performed experiments with similar results. d, RT–qPCR analysis of endogenous BRD4 mRNAs. Data are mean ± s.e.m. from three biologically independent samples. e, Annexin V/PI staining of METTL3 knockdown and control A549 cells upon JQ1 treatment (analysed by FACS). Data are from three independent experiments.

  7. Extended Data Fig. 7 Identification of a conserved alanine residue in the N-terminal region of METTL3 required for its interaction with eIF3h.

    a, Secondary structure prediction of the N-terminal (1–200) region of METTL3 protein showing putative alpha helices (blue lines). b, Evolutionary conservation of the N-terminal (1–200) region METTL3 protein. c, Computational modelling of the 3D structure of the N-terminal (77–163) region METTL3 protein, on the basis of the coordinates of RCSB Protein Data Bank entry 3HHH. d, Western blotting using the indicated antibodies. The blot is representative of two independently performed experiments with similar results. e, RT–qPCR analysis of reporter mRNAs. FLuc–MS2bs mRNA levels were normalized to RLuc mRNAs. The FLuc:RLuc ratio obtained in Flag–MS2 (control) was set to 1. Data are mean ± s.d. from six independent experiments. f, Immunoprecipitation of Flag–METTL3 (wild-type or METTL3(A155P)) and western blotting analysis using the indicated antibodies. Blots are representative of two independently performed experiments with similar results. g, Staining of recombinant protein wild-type His–Flag–MS2–METTL3 or His–Flag–MS2–METTL3(A155P). Gel is representative of two independently performed experiments with similar results.

  8. Extended Data Fig. 8 METTL3 expression correlates with lung tumour stage and promotes tumorigenicity.

    a, Staining image of control and different stages lung cancer samples (n = 75). Bottom panels show the enlarged sections of the top panels. Scale bar, 30 μM. b, Western-blotting analysis using indicated antibodies. One experiment was performed. c, d, Tumour images (c) and plot of tumour weight (d) at the end point of the xenograft experiment. Data are mean ± s.e.m. from five independent mice. P values from a two-sided t-test. e–h, Western blotting analysis using the indicated antibodies. For e, two independently were performed experiments with similar results. For f–h, one experiment was performed. i, Tumour images at the endpoint in the xenograft experiment. Scale bar, 20 mm. j, Overlapping of genes containing m6A identified in four samples from patients with lung cancer. k, Distribution of m6A sites.

  9. Extended Data Fig. 9 Polysome conformation is affected by METTL3 and m6A modification in primary human lung tumours.

    a, Electron-microscopy images of polyribosomes. Images were taken from the samples in Fig. 3e. Scale bar, 50 nm. Six independently performed experiments show similar results. b, Gene Ontology analysis. Common methylated genes refers to the methylated genes in all four patient samples. ‘Not methylated genes’ indicates genes that are not methylated in any of the four patient samples. Hypergeometric distribution (one-tail) with Bonferroni adjustment was used to determine enrichment statistical significance. c, Venn diagram showing m6A peak overlap between patient tumour samples and cells (H1299 and A549).

  10. Extended Data Fig. 10 Expression of METTL3 and eIF3h is positively correlated in many tumour types.

    a, METTL3 gene expression among TCGA tumours. b, eIF3h gene expression among TCGA tumours. a, b, Box plots display the full range of variation on the basis of the five number summaries (minimum, first quartile, median, third quartile, and maximum). NT, solid tissue normal; TP, primary solid tumour. Two-sided Wilcoxon signed-rank test was used for statistical significance. c, Plot illustrating the Pearson’s correlations of expression level between METTL3 and eIF3h in eight TCGA tumours, in which both METTL3 and eIF3h are significantly changed when compared with normal tissues.

Supplementary information

  1. Supplementary Figure

    This file contains the original source images for Western blotting and gel staining.

  2. Reporting Summary

  3. Supplementary Tables 1 and 2

    This file contains a list of primers for cloning (Supplementary Table 1) and a list of primers for qRT-PCR (Supplementary Table 2).

  4. Supplementary Table 3

    This file contains m6A peak lists for lung tumors and cancer cells.

  5. Supplementary Table 4

    This file contains RNA sequencing and polysome profiling.

  6. Supplementary Table 5

    This file contains tumor size and weight for in vivo tumor xenograft.

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