Fig. 3.

METTL3 enhances YAP mRNA translation by recruiting YTHDF1/3 and eIF3b to the translation initiation complex. a Western blot analysis of METTL3 in nuclear and cytoplasmic fractions but YTHDF1/2/3 and eIF3b only in cytoplasm fractions in A549 cells. b Schematic diagram depicting the wild-type (METTL3 WT) and catalytic mutant (METTL3 KD) of METTL3. c A549 and H1299 cells were transfected with METTL3 WT or METTL3 KD, respectively. The expressions of endogenous YAP were analyzed by qPCR. d–f The relative of m⁶A level (d, e) and mRNA level (f) of YAP were detected from A549 cells with transfection of indicated genes. g, h A549 cells were knocked down of YTHDF1, YTHDF3, and eIF3b by siRNA, then transfected with METTL3 WT or METTL3 KD, respectively. The expressions of YTHDF1, YTHDF3, and eIF3b (g) and YAP (h) were analyzed by western blot. i Co-IPs performed using lysates from A549 cells with immunoprecipitation of YTHDF1, eIF3b, eIF4E and CBP80, respectively. j Co-IPs performed using lysates from A549 cells with immunoprecipitation of METTL3, YTHDF1 and YTHDF3 antibodies, respectively. k The relative YAP enrichments were analyzed by RIP in A549 cells with immunoprecipitation of METTL3, YTHDF1, YTHDF3 and eIF3b antibodies, respectively (NC, negative control). l eIF3b interacts with the mRNA of YAP only when YTHDF1 is existed. RIP assay was carried out using YAP-WT or YAP Mut1-3 as the baits. Precipitated protein was visualized by western blotting using anti-Flag, anti-Myc, and anti-tubulin antibodies. Tubulin was detected as a control. The amounts of RNA baits used are visualized by ethidium bromide staining. Results were presented as mean ± SD of three independent experiments. **P < 0.01 indicates a significant difference between the indicated groups. NS, not significant