Fig. 6
YTHDF3 binds m6A modified GAS5 and promotes its decay. a Heatmap showing the difference transcripts between normal and YTHDF3 knockdown groups. b Meta-gene analysis depicting different m6A peaks distribution between normal and YTHDF3 knockdown groups in a normalized transcripts. c Pie chart show the different m6A peaks distribution between normal and YTHDF3 knockdown groups in all transcripts. d A Venn diagram is used to display the common differentially expressed transcripts in RNA-seq and MeRIP-seq (/FC/> 2; p-value < 0.05). e Gene ontology (GO) analysis of differentially expressed genes both in YTHDF3 knockdown and m6A peaks groups (/FC/ > 2; p-value < 0.05). f The correlation of log2 (FC) of differentially expressed transcripts in YTHDF3 knockdown cells and log2 (FC) of differentially m6A modification transcripts in YTHDF3 knockdown cells are indicated (/log FC / ≥ 1; p-value < 0.05). Group 1 (red) were those transcripts that were up-regulated in RNA-seq, whereas declined in MeRIP-seq; Group 2 (blue) were transcripts that were down-regulated in both groups (/log FC/ ≥ 1, p-value< 0.05). g IGV analysis displayed the m6A peaks among GAS5 was located in exon 8 and exon 9. h Binding motif identified by MEME visualized YTHDF3 binding motif AGGACU among GAS5. i The m6A levels of GAS5 were quantified by m6A-RNA immunoprecipitation followed by qRT–PCR in HCT116 cells treated with Control or si-YTHDF3. All experiments were performed in triplicate, and results are presented as mean ± SD. **P < 0.01 and ***P < 0.001 (j) Global m6A RNA modification treated with control or si-YTHDF3 in HCT116 cells by the m6A RNA methylation quantification analysis. All experiments were performed in triplicate, and results are presented as mean ± SD. **P < 0.01 and ***P < 0.001. k qRT-PCR of GAS5 in actinomycin D-treated CRC cells. HCT116 cells were treated with YTHDF3 plasmid, while RKO cells were treated with siRNA targeting YTHDF3. Actinomycin D (100 nM, for 8 h) was used to inhibit transcription of the indicated gene. The mean ± SD is shown for five independent experiments. ***P < 0.001. l RIP <assays for YTHDF3 were performed and the co-precipitated RNA was subjected to qRT-PCR for GAS5 (upper panel). Agarose electrophoresis of PCR products (bottom panel). Experiments were performed in triplicate, and data are presented as mean ± SD. ***P < 0.001. m RNA pulldown assays and western blot assays showed that biotinylated-GAS5 could bind YTHDF3 in CRC cells in vitro. n qRT-PCR detection of indicated genes expression. All experiments were performed in triplicate, and results are presented as mean ± SD. ***P < 0.001. o Western blots showed that silencing of YTHDF3 decreased indicated genes expression