Fig. 6: Loss of METTL3 impairs the YTHDF1-mediated translation of SPRED2.

a Metagene profiles of the m6A distribution across the transcriptome in WT and KO BMDMs. b Consensus sequence motif for m6A methylation identified in WT and KO BMDMs. c Overlap between m6A-downregulated genes (fold-change >4 and P value < 0.00001) in BMDMs and MAPK pathway-related functional genes. d Scatter plots showing m6A-related changes in cellular transcript levels in KO BMDMs versus WT BMDMs. e m6A enrichment in Spred2 mRNA in WT and KO BMDMs, as determined by m6A-RIP-qPCR. n = 3 independent experiments. f qRT-PCR (left panel) of SPRED2 expression in WT and KO BMDMs (n = 3 mice per group). The expression of SPRED2 in BMDMs (middle panel) and TAMs (right panel) was determined by immunoblotting. The blots are representative of n = 3 independent experiments. g Spred2 mRNA stability in WT and KO BMDMs treated with actinomycin D at the indicated times determined by qRT-PCR (n = 3 mice per group). h A representative polysome gradient profile for WT and KO BMDMs. i Analysis of Spred2 mRNA in the non-ribosomal fraction (<40S), 40S, 60S, 80S and polysomes of KO BMDMs compared to those of control cells. n = 3 independent experiments. j Schematic representation of mutations in the conserved coding sequence (CDS) for the investigation of the roles of m6A in SPRED2 expression. k FLAG-SPRED2-CDS WT, FLAG-SPRED2-CDS mut1, FLAG-SPRED2-CDS mut2 or FLAG-SPRED2-CDS mut1/2 was transfected into BMDMs for 24 h. Protein expression was measured by western blotting. The blots are representative of n = 3 independent experiments. l RNA immunoprecipitation (RIP) analysis of the interaction of Spred2 mRNA transcripts with YTHDF1 in WT and KO BMDMs. Enrichment of Spred2 was measured by qRT-PCR. n = 3 independent experiments. Data are means ± SD. P values were determined by hypergeometric test (b) and two-tailed t-test (c, e–g, i and l). *P ≤ 0.05 and **P < 0.01. NS (non-significant) means P > 0.05. The source data are provided as a Source data file.