Fig 2: meCLICK-Seq elucidates the relationship between m6A writers and methylation of mRNAs and lncRNAs. (a) meCLICK-Seq workflow. (b) Western blots demonstrate the extent of METTL3 and METTL16 depletion in conditional knock-down MOLM-13 cells treated with PropSeMet. Application of click-degrader does not significantly alter the levels of MTases. (c) Heat map showing decrease of methylated mRNA levels upon clicking and rescue of METTL3-dependent transcripts upon METTL3 depletion. (d) Overlap of m6A-containing mRNAs determined through m6A miCLIP and METTL3 mRNA substrates determined by meCLICK-Seq; significance indicated by Fisher’s exact test. (e) RT-qPCR-based meCLICK-Seq validation of a panel of genes, n = 3. (f) RT-qPCR-based meCLICK-Seq validation of a panel of genes in METTL3 depleted cells, n = 3. (g) Genome browser snapshot of NEAT1. Applying click machinery diminishes the WT but not METTL3 or METTL16 levels. p values determined with one-tailed t test. ns = not significant (p = 0.05). Error bars represent SD.

Fig 3: METTL3 regulates the processing of miR-196b. a–b Possible m6A methylation modification sites in miR-196b. c–d qRT-PCR analysis of miR-196b and pri-miR-196b level in CRC cells treated with siNC or siMETTL3. e–f qRT-PCR analysis of miR-196b and pri-miR-196b expression in CRC cells treated with oeNC or oeMETTL3. g–h Immunoprecipitation of endogenous METTL3. i pri-miRNAs bound to endogenous METTL3 were extracted and quantified by qRT-PCR. j m6A modification level of pri-miR-196b in CRC cells assessed by MeRIP. k m6A modification level of pri-miR-196b in SW480 cells treated with shMETTL3 detected by MeRIP. The data are presented as the mean ± SD. Three independent assays were performed (c–f) (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test)

Fig 4: Overexpression of METTL3 promoted CRC cell migration and invasion in vitro. a–b WB and qRT-PCR analysis of METTL3 expression of in CRC cells infected with oeNC or oeMETTL3. c Migration assays revealed that overexpression of METTL3 increased the migration of HCT 116 and SW480 cells. d Transwell invasion assays revealed that overexpression of METTL3 increased the invasion of HCT 116 and SW480 cells. The data are presented as the mean ± SD. Three independent assays were performed (*P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test)

Fig 5: NCBP1 increased the m6A modification of c-MYC mRNA. (A) NCBP1 affected the degree of m6A methylation, and methylene blue (MB) staining was used as a loading control. (B) c-MYC modification site in previous reports. (RNA Modification Base, http://rna.sysu.edu.cn/rmbase/). (C) c-MYC mRNA transcript high abundance of potential m6A modification. (D) The m6A abundances on c-MYC mRNA transcripts in SU-DHL-4 cells as detected by MeRIP-qPCR, which showed m6A modification in specific regions of c-MYC. Statistically significant differences are defined as follows: *P < 0.05; **P < 0.01, ***P < 0.001.