Fig. 6
Regulation of m6A-modified mRNA stability by the FBW7-YTHDF2 cascade. a Induction of the mRNA m6A levels upon YTHDF2 depletion in OVCAR8and HEY cells. The RNA m6A quantification was conducted by LC-MS/MS. [31] The YTHDF2 depletion efficiency was shown in Supplementary Fig. 9A. b Induction of the mRNA m6A levels upon FBW7 overexpression in SKOV3 and OVCAR429 cells. The FBW7 overexpression efficiency was shown in Supplementary Fig. 9B. c Decline of the mRNA m6A level caused by FBW7 depletion can be rescued by knockdown of YTHDF2. The YTHDF2 depletion and FBW7 overexpression efficiency was shown in Supplementary Fig. 9C. d RNA-sequencing analysis reveals a distinct gene expression patterns in response to YTHDF2 depletion in SKOV3 cells. e Distribution of m6A modification in mRNA transcripts. The m6A signal is mostly enriched in the coding sequences. f The m6A consensus sequence motif is identified in ovarian cancer cells. g Integration of the RNA-Sequencing and m6A-Sequencing results revealed 25 genes with 39 peaks are consistently elevated upon YTHDF2 knockdown, which are displayed in Supplementary table 4 (h). The m6A modification site of BMF mRNA was in the 3′-UTR region. i Knockdown of YTHDF2 increases BMF mRNA level in SKOV3 cells. j Knockdown of YTHDF2 prolongs the half-life of BMF mRNA. Cells were treated with actinomycin D (5 μg/ml) for 3 or 6 h followed by RT-qPCR analysis. k The mRNA level of BMF is elevated upon the treatment of 50 μM 3-deazaadenosine (DAA) in SKOV3 cells. l The interaction between YTHDF2 and BMF pre-mRNA was detected by the RIP assay. m The mRNA level of BMF is significantly elevated in SKOV3 cells stably overexpressing FBW7. n The relative m6A level of BMF pre-mRNA was detected by MeRIP-qPCR in SKOV3 cells. o The FBW7-mediated regulation of BMF is dependent on YTHDF2 demonstrated by qRT-PCR detection. p YTHDF2 is inversely correlated with BMF expression in 64 epithelial ovarian cancer tissues. q FBW7 is positively correlated with BMF expression in 64 epithelial ovarian cancer tissues. r The working model for the regulation of m6A-dependent turnover of BMF mRNA by the FBW7-YTHDF2 cascade. Results were presented as mean ± SD of three independent experiments. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups