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. 2020 Apr 3;19:72. doi: 10.1186/s12943-020-01190-w

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Transcriptome-wide m⁶A-seq and RNA-seq assays identified potential targets of METTL3 in colorectal cancer. (a) The m⁶A dot blot assay of global m⁶A abundance in mRNA of HCT116 WT and METTL3-knockout cells. MB, methylene blue staining (as a loading control). (b) The global m⁶A levels in mRNA of HCT116 WT and METTL3- knockout cells were measured by the EpiQuik™ m⁶A RNA Methylation Quantification Kit, n = 3, nonparametric Mann–Whitney test. (c) LC-MS/MS quantification of the m⁶A/A ratio in poly(A) RNA in HCT116 WT and METTL3-KO cells. (d) HOMER motif analysis revealed the top consensus m⁶A motif in HCT116 cells. (e) Graphs of m⁶A peak distribution illustrating the proportion of common m⁶A peaks in the indicated regions in HCT116 cells. (f) Cumulative curves of m⁶A abundance (log₂(m⁶A EF + 1)) in HCT116 WT and METTL3-KO cells. The abundance of m⁶A immunoprecipitation was normalized to input when calculating the enrichment fold. P value was calculated using two-sided Wilcoxon and Mann–Whitney test. EF, enrichment folds. (g) Circos plot showing the distribution of hypermethylated (hyper) and hypomethylated (hypo) m⁶A peaks in the human transcriptome of HCT116 METTL3-KO cells compared with WT cells. (h) Distribution of genes with a significant change in both m⁶A level and gene expression level in HCT116 METTL3-KO cells compared with WT cells. (i) The flow chart for selected candidate target genes of METTL3 in HCT116 METTL3-KO cells is shown. (j) The relative abundance of m⁶A sites along HK2 and SLC2A1 (GLUT1) mRNA in HCT116 WT and METTL3-KO cells, as detected by m⁶A-seq. The orange rectangles indicated that the m⁶A peaks had a significantly decreased abundance. (k) Agarose electrophoresis and real-time PCR analysis of MeRIP assays in CRC cells showing the direct binding between the m⁶A antibody and HK2 5’UTR, HK2 3’UTR and SLC2A1 (GLUT1) 3’UTR. (l) MeRIP-qPCR analysis of HK2 5’UTR, HK2 3’UTR and SLC2A1 (GLUT1) 3’UTR m⁶A levels in HCT116 WT and METTL3-KO cells, n = 3, nonparametric Mann–Whitney test. (WT, wild type; METTL3-KO, METTL3-knockout)