Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation (original) (raw)

Change history

In the version of this Article originally published, the authors incorrectly listed an accession code as GES90642. The correct code is GSE90642. This has now been amended in all online versions of the Article.

A Correction to this paper has been published: https://doi.org/10.1038/s41556-020-00580-y

References

  1. Jia, G. et al. N 6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).
    Article PubMed PubMed Central CAS Google Scholar
  2. Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).
    Article PubMed CAS Google Scholar
  3. Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).
    PubMed PubMed Central CAS Google Scholar
  4. Zhao, X. et al. FTO-dependent demethylation of N 6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 24, 1403–1419 (2014).
    Article PubMed PubMed Central CAS Google Scholar
  5. Su, R. et al. R-2HG exhibits anti-tumor activity by targeting FTO/m6A/MYC/CEBPA signaling. Cell 72, 90–105 (2018).
    Article CAS Google Scholar
  6. Liu, N. et al. N 6-methyladenosine-dependent RNA structural switches regulate RNA–protein interactions. Nature 518, 560–564 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  7. Geula, S. et al. Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347, 1002–1006 (2015).
    Article PubMed CAS Google Scholar
  8. Wang, Y. et al. N 6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat. Cell Biol. 16, 191–198 (2014).
    Article PubMed PubMed Central CAS Google Scholar
  9. Chen, T. et al. m6A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. Cell Stem Cell 16, 289–301 (2015).
    Article PubMed CAS Google Scholar
  10. Wang, X. et al. N 6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).
    Article PubMed CAS Google Scholar
  11. Zhao, B. S., Roundtree, I. A. & He, C. Post-transcriptional gene regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42 (2016).
    Article PubMed PubMed Central CAS Google Scholar
  12. Li, Z. et al. FTO plays an oncogenic role in acute myeloid leukemia as a N 6-methyladenosine RNA demethylase. Cancer Cell 31, 127–141 (2017).
    Article PubMed CAS Google Scholar
  13. Zhao, B. S. et al. m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition. Nature 542, 475–478 (2017).
    Article PubMed PubMed Central CAS Google Scholar
  14. Wang, X. et al. N 6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  15. Du, H. et al. YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4–NOT deadenylase complex. Nat. Commun. 7, 12626 (2016).
    Article PubMed PubMed Central CAS Google Scholar
  16. Xiao, W. et al. Nuclear m6A reader YTHDC1 regulates mRNA splicing. Mol. Cell 61, 507–519 (2016).
    Article CAS PubMed Google Scholar
  17. Bell, J. L. et al. Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): post-transcriptional drivers of cancer progression? Cell. Mol. Life Sci. 70, 2657–2675 (2013).
    Article PubMed CAS Google Scholar
  18. Nielsen, J. et al. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Mol. Cell. Biol. 19, 1262–1270 (1999).
    Article PubMed PubMed Central CAS Google Scholar
  19. Noubissi, F. K. et al. CRD-BP mediates stabilization of βTrCP1 and c-myc mRNA in response to β-catenin signalling. Nature 441, 898–901 (2006).
    Article PubMed CAS Google Scholar
  20. Huttelmaier, S. et al. Spatial regulation of β-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438, 512–515 (2005).
    Article PubMed CAS Google Scholar
  21. Weidensdorfer, D. et al. Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs. RNA 15, 104–115 (2009).
    Article PubMed PubMed Central CAS Google Scholar
  22. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).
    Article PubMed PubMed Central CAS Google Scholar
  23. Behm-Ansmant, I., Gatfield, D., Rehwinkel, J., Hilgers, V. & Izaurralde, E. A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay. EMBO J. 26, 1591–1601 (2007).
    Article PubMed PubMed Central CAS Google Scholar
  24. Mangus, D. A., Evans, M. C. & Jacobson, A. Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol. 4, 223 (2003).
    Article PubMed PubMed Central Google Scholar
  25. Fan, X. C. & Steitz, J. A. Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs. EMBO J. 17, 3448–3460 (1998).
    Article PubMed PubMed Central CAS Google Scholar
  26. Salton, M. et al. Matrin 3 binds and stabilizes mRNA. PLoS ONE 6, e23882 (2011).
    Article PubMed PubMed Central CAS Google Scholar
  27. Boudoukha, S., Cuvellier, S. & Polesskaya, A. Role of the RNA-binding protein IMP-2 in muscle cell motility. Mol. Cell. Biol. 30, 5710–5725 (2010).
    Article PubMed PubMed Central CAS Google Scholar
  28. Wachter, K., Kohn, M., Stohr, N. & Huttelmaier, S. Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains. Biol. Chem. 394, 1077–1090 (2013).
    Article PubMed CAS Google Scholar
  29. Stohr, N. et al. ZBP1 regulates mRNA stability during cellular stress. J. Cell Biol. 175, 527–534 (2006).
    Article PubMed PubMed Central CAS Google Scholar
  30. Doyle, G. A. et al. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins. Nucleic Acids Res. 26, 5036–5044 (1998).
    Article PubMed PubMed Central CAS Google Scholar
  31. Bley, N. et al. Stress granules are dispensable for mRNA stabilization during cellular stress. Nucleic Acids Res. 43, e26 (2015).
    Article PubMed CAS Google Scholar
  32. Valverde, R., Edwards, L. & Regan, L. Structure and function of KH domains. FEBS J. 275, 2712–2726 (2008).
    Article PubMed CAS Google Scholar
  33. Nielsen, J., Kristensen, M. A., Willemoes, M., Nielsen, F. C. & Christiansen, J. Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability. Nucleic Acids Res. 32, 4368–4376 (2004).
    Article PubMed PubMed Central CAS Google Scholar
  34. Chao, J. A. et al. ZBP1 recognition of β-actin zipcode induces RNA looping. Genes Dev. 24, 148–158 (2010).
    Article PubMed PubMed Central CAS Google Scholar
  35. Dai, N. et al. mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry. Genes Dev. 25, 1159–1172 (2011).
    Article PubMed PubMed Central CAS Google Scholar
  36. Roundtree, I. A., Evans, M. E., Pan, T. & He, C. Dynamic RNA modifications in gene expression regulation. Cell 169, 1187–1200 (2017).
  37. Peng, S. S., Chen, C. Y., Xu, N. & Shyu, A. B. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein. EMBO J. 17, 3461–3470 (1998).
    Article PubMed PubMed Central CAS Google Scholar
  38. Brengues, M., Teixeira, D. & Parker, R. Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486–489 (2005).
    Article PubMed PubMed Central CAS Google Scholar
  39. Huch, S. et al. The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae. Biol. Open 5, 1388–1399 (2016).
    Article PubMed PubMed Central CAS Google Scholar
  40. Bett, J. S. et al. The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability. Biochem. J. 451, 185–194 (2013).
    Article PubMed CAS Google Scholar
  41. Weng, H. et al. METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m6A modification. Cell Stem Cell 22, 191–205 (2018).
  42. Sun, W. J. et al. RMBase: a resource for decoding the landscape of RNA modifications from high-throughput sequencing data. Nucleic Acids Res. 44, 259–265 (2016).
    Article CAS Google Scholar
  43. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime _cis_-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).
    Article PubMed PubMed Central CAS Google Scholar
  44. Rinn, J. L. et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323 (2007).
    Article PubMed PubMed Central CAS Google Scholar
  45. Chen, C. Y. A., Ezzeddine, N. & Shyu, A. B. Messenger RNA half-life measurements in mammalian cells. Methods Enzymol. 448, 335–357 (2008).
    Article PubMed PubMed Central CAS Google Scholar
  46. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
    Article PubMed PubMed Central Google Scholar
  47. Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).
    Article PubMed PubMed Central CAS Google Scholar
  48. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10–12 (2011).
    Article Google Scholar
  49. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
    Article PubMed PubMed Central CAS Google Scholar
  50. Corcoran, D. L. et al. PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol. 12, R79 (2011).
    Article PubMed PubMed Central CAS Google Scholar
  51. Zisoulis, D. G. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 17, 173–179 (2010).
    Article PubMed PubMed Central CAS Google Scholar
  52. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  53. Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
    Article PubMed CAS Google Scholar
  54. Meng, J. et al. A protocol for RNA methylation differential analysis with MeRIP-seq data and exomePeak R/Bioconductor package. Methods 69, 274–281 (2014).
    Article PubMed PubMed Central CAS Google Scholar
  55. Weng, H. Y. et al. Inhibition of miR-17 and miR-20a by oridonin triggers apoptosis and reverses chemoresistance by derepressing BIM-S. Cancer Res. 74, 4409–4419 (2014).
    Article PubMed CAS Google Scholar
  56. Eismann, T. et al. Peroxiredoxin-6 protects against mitochondrial dysfunction and liver injury during ischemia–reperfusion in mice. Am. J. Physiol. Gastr. L. 296, 266–274 (2009).
    Article CAS Google Scholar
  57. Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
    Article PubMed PubMed Central CAS Google Scholar
  58. Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).
    Article PubMed CAS Google Scholar

Download references

Acknowledgements

We thank the Proteomics Laboratory at the University of Cincinnati for mass spectrometry analysis; the Transgenic Animal and Genome Editing Core at the Cincinnati Children’s Hospital Medical Center for design and construction of sgRNA vectors; the Genomics, Epigenomics and Sequencing Core at the University of Cincinnati and the Genomic Facility at the University of Chicago for next-generation sequencing. This work was supported in part by the National Institutes of Health (NIH) R01 grants CA214965 (J.C.), CA211614 (J.C.), CA178454 (J.C.), CA182528 (J.C.), CA163493 (J.-L.G.), RM1 HG008935 (C.He), 1S10RR027015-01 (K.D.G.) and grants 2017YFA0504400 (J.Y.), 91440110 (J.Y.) and 31671349 (L.Q.) from the National Nature Science Foundation of China. J.C. is a Leukemia & Lymphoma Society (LLS) Scholar. C.He is an investigator of the Howard Hughes Medical Institute (HHMI). B.S.Z. is an HHMI International Student Research Fellow.

Author information

Author notes

  1. These authors contributed equally: Huilin Huang, Hengyou Weng, Wenju Sun, Xi Qin and Hailing Shi.

Authors and Affiliations

  1. Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA
    Huilin Huang, Hengyou Weng, Xi Qin, Huizhe Wu, Ana Mesquita, Jennifer R. Skibbe, Rui Su, Xiaolan Deng, Lei Dong, Chenying Li, Yungui Wang, Chao Hu, Kyle Ferchen, Kenneth D. Greis, Xi Jiang, Jun-Lin Guan & Jianjun Chen
  2. Department of Systems Biology, City of Hope, Monrovia, CA, USA
    Huilin Huang, Hengyou Weng, Xi Qin, Huizhe Wu, Rui Su, Xiaolan Deng, Lei Dong, Chenying Li, Xi Jiang & Jianjun Chen
  3. Key Laboratory of Gene Engineering of the Ministry of Education, Sun Yat-sen University, Guangzhou, China
    Wenju Sun, Lianghu Qu & Jianhua Yang
  4. State Key Laboratory for Biocontrol, Sun Yat-sen University, Guangzhou, China
    Wenju Sun, Lianghu Qu & Jianhua Yang
  5. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA
    Hailing Shi, Boxuan Simen Zhao, Chang Liu, Sigrid Nachtergaele & Chuan He
  6. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA
    Hailing Shi, Boxuan Simen Zhao, Chang Liu, Sigrid Nachtergaele & Chuan He
  7. Department of Pharmacology, School of Pharmacy, China Medical University, Shenyang, China
    Huizhe Wu, Xiaolan Deng & Minjie Wei
  8. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
    Celvie L. Yuan & Yueh-Chiang Hu
  9. Institute of Molecular Medicine, Department of Molecular Cell Biology, Martin Luther University, Halle, Germany
    Stefan Hüttelmaier
  10. Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
    Miao Sun
  11. Key Laboratory of Hematopoietic Malignancies, Department of Hematology, The First Affiliated Hospital of Zhejiang University, Hangzhou, China
    Chenying Li, Yungui Wang & Chao Hu

Authors

  1. Huilin Huang
    You can also search for this author inPubMed Google Scholar
  2. Hengyou Weng
    You can also search for this author inPubMed Google Scholar
  3. Wenju Sun
    You can also search for this author inPubMed Google Scholar
  4. Xi Qin
    You can also search for this author inPubMed Google Scholar
  5. Hailing Shi
    You can also search for this author inPubMed Google Scholar
  6. Huizhe Wu
    You can also search for this author inPubMed Google Scholar
  7. Boxuan Simen Zhao
    You can also search for this author inPubMed Google Scholar
  8. Ana Mesquita
    You can also search for this author inPubMed Google Scholar
  9. Chang Liu
    You can also search for this author inPubMed Google Scholar
  10. Celvie L. Yuan
    You can also search for this author inPubMed Google Scholar
  11. Yueh-Chiang Hu
    You can also search for this author inPubMed Google Scholar
  12. Stefan Hüttelmaier
    You can also search for this author inPubMed Google Scholar
  13. Jennifer R. Skibbe
    You can also search for this author inPubMed Google Scholar
  14. Rui Su
    You can also search for this author inPubMed Google Scholar
  15. Xiaolan Deng
    You can also search for this author inPubMed Google Scholar
  16. Lei Dong
    You can also search for this author inPubMed Google Scholar
  17. Miao Sun
    You can also search for this author inPubMed Google Scholar
  18. Chenying Li
    You can also search for this author inPubMed Google Scholar
  19. Sigrid Nachtergaele
    You can also search for this author inPubMed Google Scholar
  20. Yungui Wang
    You can also search for this author inPubMed Google Scholar
  21. Chao Hu
    You can also search for this author inPubMed Google Scholar
  22. Kyle Ferchen
    You can also search for this author inPubMed Google Scholar
  23. Kenneth D. Greis
    You can also search for this author inPubMed Google Scholar
  24. Xi Jiang
    You can also search for this author inPubMed Google Scholar
  25. Minjie Wei
    You can also search for this author inPubMed Google Scholar
  26. Lianghu Qu
    You can also search for this author inPubMed Google Scholar
  27. Jun-Lin Guan
    You can also search for this author inPubMed Google Scholar
  28. Chuan He
    You can also search for this author inPubMed Google Scholar
  29. Jianhua Yang
    You can also search for this author inPubMed Google Scholar
  30. Jianjun Chen
    You can also search for this author inPubMed Google Scholar

Contributions

H.H., H.Weng and J.C. conceived and designed the entire project. H.H., H.Weng, C.He, J.Y. and J.C. designed and supervised the research. H.H., H.Weng, X.Q., H.S., H.Wu, B.S.Z., A.M., C.Liu, C.L.Y., J.R.S., R.S., X.D., M.S., C.Li, S.N., Y.W., C.Hu, K.F. and J.C. performed the experiments and/or data analyses. H.H., H.Weng, W.S., L.D. and J.Y. performed the genome-wide or transcriptome-wide data analyses. Y.-C.H., S.H., K.D.G., X.J., M.W., L.Q., J.-L.G., C.He, J.Y. and J.C. contributed reagents/analytic tools and/or grant support. H.H., H.Weng, W.S., H.S., B.S.Z., A.M., S.N., C.He, J.Y. and J.C. wrote and revised the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence toChuan He, Jianhua Yang or Jianjun Chen.

Ethics declarations

Competing interests

C.He is a scientific founder of Accent Therapeutics, Inc.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Selective binding of IGF2BP proteins to m6A methylated RNA.

(a) Dot blot confirming the m6A modification of single strand (ss) RNA probes used in RNA pulldown assay. Note that ss-A probe without m6A modification has no m6A signal. Methylene blue (MB) staining served as a loading control. (b) In vitro binding of RNA probes (0-1.0µM) with IGF2BP proteins from HEK293T nuclear extract. The gray signal of bands in Western blots (upper) was quantified by Image Master Total Lab and is shown below. (c) In vitro binding of denatured (heated at 99 °C for 10 min) ssRNA probes with endogenous IGF2BP proteins under conditions that prevent (on ice, ICE) or allow (at room temperature, RT) RNA refolding. (d) In vitro binding of m6A-methylated or unmethylated RNA probes with 0.5 µg of recombinant IGF2BP1or IGF2BP2 protein or 1.0 µg of recombinant IGF2BP3 protein produced from HEK293T cells. (e) Gel shift assays measuring the dissociation constant (_K_d, nM) of recombinant IGF2BP proteins with methylated (ss-m6A) or unmethylated (ss-A) ssRNA Probes. (f) Sequences and modifications of hairpin RNA probes used in RNA pulldown assay. Note that hp-A probe without m6A modification has no m6A signal. (g) Specific binding of IGF2BP proteins from HEK293T nuclear extract with methylated ssRNA and hpRNA probes as detected by RNA pulldown and Western blot. (h) Numbers of m6A modifications within binding sites of RBPs. The three IGF2BP paralogues were shown in red. Analyses were performed twice with similar results. (i and j) Enrichment of m6A in IGF2BP-bound RNA. m6A methylation of mRNP complexes isolated from HEK293T cells with ectopic expression of FLAG-IGF2BP1-3 (i) or from parental HepG2 cells (j) was evaluated by dot blot. (k and l) Consensus sequences of binding sites of endogenous IGF2BPs in HepG2 cells (k) and hESCs (l) detected by HOMER Motif analysis with ENCODE eCLIP data. Images of dot blot or western blot in a, b, c, d, e, f, g, i, j were representative of 3 independent experiments. Unprocessed scans of western blot analysis are available in Supplementary Figure 8.

Supplementary Figure 2 Functional annotation of IGF2BP targets.

(a) Western blot showing downregulation of IGF2BPs in HepG2 cells infected with lentiviral shRNAs against each IGF2BP, representative of 3 independent experiments. GAPDH was used as a loading control. Cells transduced with non-specific control (NS) or #1 shRNA for each IGF2BP were used for RNA-seq and mRNA stability profiling. (b) Enrichment plots of CLIP+RIP targets of IGF2BPs. NES, normalized enrichment score; FDR, false discovery rate. Note that FDR <0.25 was considered significant in gene set enrichment analysis (GSEA) analysis. (c) Representative biological processes and KEGG pathways in which IGF2BP downregulated targets are enriched. (d) GSEA analysis of shared downregulated targets by knockdown of IGF2BP1, IGF2BP2, and IGF2BP3. (e) Changes of MYC and FSCN1 mRNA levels in IGF2BP1 or/and YTHDF2 knockdown Hela cells. Values are mean±s.d. of n =3 independent experiments, and two-tailed student _t_-test were used (***, P <0.001). (f) Cumulative frequency of mRNA log2-fold change showing global reduction of METTL14 target genes upon IGF2BP silencing. P values were calculated using two-sided Wilcoxon and Mann-Whitney test. Unprocessed scans of western blot analysis are available in Supplementary Figure 8. Source data of e can be found in Supplementary Table 3.

Supplementary Figure 3 IGF2BPs regulate mRNA stability.

(a) Cumulative distribution of mRNA half-life in non-target or YTHDF2 target genes in HepG2 and Hela cells. (b) Distribution of mRNA half-lives in IGF2BP3 CLIP targets in HepG2 cells with shIGF2BP3 or shNS. (c) Cumulative distribution of mRNA half-life in IGF2BP3 CLIP targets in shIGF2BP3 or shNS HepG2 cells. mRNA half-life analyses in a, b, c were repeated twice. (d) mRNA stability assay showing decreased mRNA half-lives of FSCN1, TK1 and MARCKSL1 upon knockdown of IGF2BPs in HepG2 cells. (e) mRNA stability assay showing decreased mRNA half-lives of MYC, FSCN1, TK1 and MARCKSL1 upon knockdown of IGF2BPs in human cord blood CD34+ cells. (f) mRNA stability assay showing decreased mRNA half-lives of FSCN1, TK1 and MARCKSL1 upon knockdown of METTL3 or METTL14 in HepG2 cells. (g and h) Colocalization of IGF2BP proteins with stress granule marker TIAR (g) or P-body marker DCP1A (h) in Hela cells after heat shock at 42 ˚C for 1 hour. Images were representative of 3 independent experiments. Arrows indicate colocalization in cytoplasmic granules. Scale bar=10µm. P values were calculated using two-sided Wilcoxon and Mann-Whitney test in a, b, c. Values are mean±s.d. of n =3 independent experiments, and exponential regression was used in d, e, f. Source data of d, e, f can be found in Supplementary Table 3.

Supplementary Figure 4 IGF2BPs facilitate mRNA translation.

(a) HEK293T cells were transfected with FLAG-IGF2BPs and lysed. Polysome-fractionated samples were grouped to non-ribosome mRNPs, 40S-80S (translatable) and polysome (actively translating), and analyzed by Western blot using antibodies against FLAG, HuR, and the translation initiation factor eIF3 core subunits eIF3A and eIF3B. (b) Distribution of endogenous IGF2BP proteins in polysome fractions of HepG2 cells. (c) Polysomal profiling of endogenous MYC mRNA in IGF2BP1 knockdown or control HEK293T cells. Values are mean±s.d. of n =2 independent experiments. (d) Hela cells were heat shocked at 42 °C for 1 hour and allowed to recover for different time period. Polysomal profiling was performed to detect endogenous IGF2BP2 during heat shock recovery. Results of a, b and d are representative of 2 independent experiments. Unprocessed scans of western blot analysis are available in Supplementary Figure 8. Source data of c can be found in Supplementary Table 3.

Supplementary Figure 5 Mechanism by which IGF2BP recognizes their target mRNAs and inhibits target expression.

(a) RIP-qPCR showing association of endogenous IGF2BP1 or IGF2BP2 with MYC CRD in HepG2 cells. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s _t_-tests were used (***, P <0.001;). (b) Schematic diagram of CRD-wt and CRD-mut firefly luciferase reporters. The 249-nt DNA sequence of wild-type CRD was inserted at the XhoI site ahead of the stop codon of firefly luciferase gene in pMIR-REPORT vector to give rise to the CRD-WT reporter. For the CRD-mut reporter, A-T substitutions (shown in red) were made within m6A consensus (in grey background). Note that only part of the CRD sequence that contains mutation sites is shown. The sequences of CRD RNA oligos used for in vitro binding assay were shown in blue below the sequences of CRD insertions. Arrows indicate locations of primers used in RIP-qPCR assays. The primers were designed to distinguish expression of MYC-CRD reporters from endogenous MYC mRNA. (c) RNA pulldown assay showing in vitro binding of IGF2BPs to mutated (A to U mutations, labeled as U) and m6A methylated (labeled as m6A) CRD RNA probes, representative of 3 independent experiments. (d) Relative luciferase activity of CRD-wt reporter when cotransfected with indicated amount of IGF2BPs expression vectors. HEK293T cells and Hela cells were examined 24 hours and 48 hours after transfection, respectively. Values are mean of n =2 independent experiments. (e and f) RNA pulldown assays showing in vitro binding of IGF2BP2 variants to ssRNA oligos (e) or hpRNA oligos (f), representative of 3 independent experiments. Unprocessed scans of western blot analysis are available in Supplementary Figure 8. Source data of a and d can be found in Supplementary Table 3.

Supplementary Figure 6 MYC is an important oncogenic target of IGF2BPs.

(a and b) Dysregulation of IGF2BPs in human cancers. Data from The Cancer Genome Atlas (TCGA) was analyzed and shown for cross-cancer alteration (a) and RNA expression (b) of IGF2BPs using cBioPortal (www.cbioportal.org). (c) Representative images of 3 independent experiments showing the effect of IGF2BP knockdown on cell migration and invasion. (d and e) Quantification of wound closure (d) and representative images (e) at the indicated time points in IGF2BPs-silenced and control Hela cells. Values are mean±s.d. of n =3 independent experiments. (f) qPCR confirmed the knockdown of MYC by siRNA at 72 hours after transfection. Values are mean±s.d. of n =3 independent experiments. (g) Effect of MYC siRNA on cell proliferation as assessed by MTT assays. Values are mean±s.d. of n =3 independent experiments. (h) Representative images (left) and colony numbers (right) of Hela and HepG2 cells transfected with control (siNC) or MYC siRNA (siMYC). Colonies were counted from 3 replicate wells of 2 independent experiments. (i and j) Effect of MYC siRNA on cell migration and invasion examined by transwell assays in Hela (i) and HepG2 cells (j). Numbers of migrated and invaded cells were counted from 3 independent experiments. P values were calculated using two-tailed student’s t-test in d, f and g (**, P <0.01; ***, P <0.001). Unprocessed scans of western blot analysis are available in Supplementary Figure 8. Source data of d, f, g, h, i, j can be found in Supplementary Table 3.

Supplementary Figure 7 Comparison of IGF2BPs and YTHDF2 binding sites.

(a) Venn diagram showing the overlapping of IGF2BPs and YTHDF2 binding sites. The binding sites of IGF2BPs and YTHDF2 were called by using PARalyzer software with stringent parameter (Minimum number of reads=5 and minimum read depth=5). (b) Significant motifs within YTHDF2 binding sites by HomerMotif analysis of all significant peaks of YTHDF2 Analyses in a and b were performed twice with similar results. (c) Box plots showing GC content of total (upper) or GGAC-containing binding sites (lower) of IGF2BPs and YTHDF2. The minima, maxima, centre, percentiles and n number were shown. (d) Cumulative curves showing GC content of total (upper) or GGAC-containing binding sites (lower) of IGF2BPs and YTHDF2. P values were calculated using two-sided Wilcoxon and Mann-Whitney test. PAR-CLIP data analyzed in c and d were from 3 biological independent experiments.

Supplementary Figure 8 Unprocessed gel blots.

Of note, for some immunoblotting assays membranes were cut into several pieces to incubate with different antibodies, and therefore the raw images of these membranes are of small size.

Supplementary information

Rights and permissions

About this article

Cite this article

Huang, H., Weng, H., Sun, W. et al. Recognition of RNA _N_6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.Nat Cell Biol 20, 285–295 (2018). https://doi.org/10.1038/s41556-018-0045-z

Download citation