Fig. 6: YTHDF3 recognizes m6A modification sites and interacts with degradation-related proteins participating in the regulation of IFITM1.
a, Left: YTHDF3 knockdown was performed in NP460, NP460-EBV, HK1 and HK1-EBV cells; representative of 2 independent experiments. Middle: DEGs were identified through RNA-seq. Right: Venn diagram showing the overlap between DEGs and m6A modification sites of 7,104 human genes reported in ref. 42. Seven DEGs including IFITM1 were screened out. b, Fold enrichment of IFITM1 as determined by RIP−qPCR. c, Left: MeRIP-sequencing, grey region shows m6A enrichment based on the input RNA. m6A motif sequences corresponding to the immunoprecipitated enriched region are indicated by different colours in different cells (NP69, green; HK1, red; HK1-shYTHDF3, magenta; input, grey). The RNA sequence at the bottom is the predicted m6A site binding with YTHDF3. Right: MeRIP−qPCR, fold enrichment of IFITM1 as determined by MeRIP−qPCR. d, Left: silver stain of the eluted protein from tandem affinity purification and mass spectrometry. Lines 1, 2 and 3 show the purified proteins from HK1-TAP, HK1-TAP-YTHDF3WT and HK1-TAP-YTHDF3DM, respectively. Right: Venn diagram of the exogenous YTHDF3-binding proteins (HK1-YTHDF3-Exo-TAP: from purified proteins in HK1-TAP-YTHDF3WT) and endogenous YTHDF3-binding proteins (HK1-YTHDF3-Endo-TAP: from endogenous YTHDF3-IP). The red rectangle serves to differentiate the two bands on the gel more clearly. e, Co-IP assays validated that endogenous YTHDF3 co-immunoprecipitated with DDX5, DDX6 and DDX17. HK1 cell lysates were immunoprecipitated (IP) with YTHDF3 antibody, followed by an immunoblotting (IB) assay with the corresponding DDX antibodies. IgG-IP samples were included as a control. f, Lentiviruses encoding YTHDF3 overexpression or DDX5 knockdown and their corresponding negative control lentiviruses (vector + shLacZ) were transfected into HK1 cells, and immunoblotting assays were performed using anti-YTHDF3, DDX5 and IFITM1. Representative of 3 independent experiments (d–f). g, IFITM1 remaining, detected by RT−qPCR after treating with transcriptional inhibitor ActD. h, Cells from f were exposed to EBV-GFP for 3 h, EBV copy numbers were measured by TaqMan−qPCR and results from control groups were taken as 100%. Mean ± s.e.m., n = 3 independent experiments, *P < 0.05, **P < 0.01, two-tailed t-test (b,c,g,h).
