Abstract

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.

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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.

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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.

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

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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.

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.

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https://doi.org/10.1038/s41556-018-0045-z