N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

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

Change history

  • 07 June 2018

    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.


  1. 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).

  2. 2.

    Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

  3. 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).

  4. 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).

  5. 5.

    Su, R. et al. R-2HG exhibits anti-tumor activity by targeting FTO/m6A/MYC/CEBPA signaling. Cell 72, 90–105 (2018).

  6. 6.

    Liu, N. et al. N 6-methyladenosine-dependent RNA structural switches regulate RNA–protein interactions. Nature 518, 560–564 (2015).

  7. 7.

    Geula, S. et al. Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347, 1002–1006 (2015).

  8. 8.

    Wang, Y. et al. N 6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat. Cell Biol. 16, 191–198 (2014).

  9. 9.

    Chen, T. et al. m6A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. Cell Stem Cell 16, 289–301 (2015).

  10. 10.

    Wang, X. et al. N 6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

  11. 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).

  12. 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).

  13. 13.

    Zhao, B. S. et al. m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition. Nature 542, 475–478 (2017).

  14. 14.

    Wang, X. et al. N 6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

  15. 15.

    Du, H. et al. YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4–NOT deadenylase complex. Nat. Commun. 7, 12626 (2016).

  16. 16.

    Xiao, W. et al. Nuclear m6A reader YTHDC1 regulates mRNA splicing. Mol. Cell 61, 507–519 (2016).

  17. 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).

  18. 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).

  19. 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).

  20. 20.

    Huttelmaier, S. et al. Spatial regulation of β-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438, 512–515 (2005).

  21. 21.

    Weidensdorfer, D. et al. Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs. RNA 15, 104–115 (2009).

  22. 22.

    Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

  23. 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).

  24. 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).

  25. 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).

  26. 26.

    Salton, M. et al. Matrin 3 binds and stabilizes mRNA. PLoS ONE 6, e23882 (2011).

  27. 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).

  28. 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).

  29. 29.

    Stohr, N. et al. ZBP1 regulates mRNA stability during cellular stress. J. Cell Biol. 175, 527–534 (2006).

  30. 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).

  31. 31.

    Bley, N. et al. Stress granules are dispensable for mRNA stabilization during cellular stress. Nucleic Acids Res. 43, e26 (2015).

  32. 32.

    Valverde, R., Edwards, L. & Regan, L. Structure and function of KH domains. FEBS J. 275, 2712–2726 (2008).

  33. 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).

  34. 34.

    Chao, J. A. et al. ZBP1 recognition of β-actin zipcode induces RNA looping. Genes Dev. 24, 148–158 (2010).

  35. 35.

    Dai, N. et al. mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry. Genes Dev. 25, 1159–1172 (2011).

  36. 36.

    Roundtree, I. A., Evans, M. E., Pan, T. & He, C. Dynamic RNA modifications in gene expression regulation. Cell 169, 1187–1200 (2017).

  37. 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).

  38. 38.

    Brengues, M., Teixeira, D. & Parker, R. Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486–489 (2005).

  39. 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).

  40. 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).

  41. 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. 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).

  43. 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).

  44. 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).

  45. 45.

    Chen, C. Y. A., Ezzeddine, N. & Shyu, A. B. Messenger RNA half-life measurements in mammalian cells. Methods Enzymol. 448, 335–357 (2008).

  46. 46.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

  47. 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).

  48. 48.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10–12 (2011).

  49. 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).

  50. 50.

    Corcoran, D. L. et al. PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol. 12, R79 (2011).

  51. 51.

    Zisoulis, D. G. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 17, 173–179 (2010).

  52. 52.

    Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).

  53. 53.

    Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

  54. 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).

  55. 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).

  56. 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).

  57. 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).

  58. 58.

    Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

Download references


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.


  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


  1. Search for Huilin Huang in:

  2. Search for Hengyou Weng in:

  3. Search for Wenju Sun in:

  4. Search for Xi Qin in:

  5. Search for Hailing Shi in:

  6. Search for Huizhe Wu in:

  7. Search for Boxuan Simen Zhao in:

  8. Search for Ana Mesquita in:

  9. Search for Chang Liu in:

  10. Search for Celvie L. Yuan in:

  11. Search for Yueh-Chiang Hu in:

  12. Search for Stefan Hüttelmaier in:

  13. Search for Jennifer R. Skibbe in:

  14. Search for Rui Su in:

  15. Search for Xiaolan Deng in:

  16. Search for Lei Dong in:

  17. Search for Miao Sun in:

  18. Search for Chenying Li in:

  19. Search for Sigrid Nachtergaele in:

  20. Search for Yungui Wang in:

  21. Search for Chao Hu in:

  22. Search for Kyle Ferchen in:

  23. Search for Kenneth D. Greis in:

  24. Search for Xi Jiang in:

  25. Search for Minjie Wei in:

  26. Search for Lianghu Qu in:

  27. Search for Jun-Lin Guan in:

  28. Search for Chuan He in:

  29. Search for Jianhua Yang in:

  30. Search for Jianjun Chen in:


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.

Competing interests

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

Corresponding authors

Correspondence to Chuan He or Jianhua Yang or Jianjun Chen.

Integrated supplementary information

  1. 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 (Kd, 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.

  2. 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.

  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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

About this article

Publication history






Further reading