Fig. 5: “Writer” NOP2 and “Reader” ALYREF promote the stability of EZH2 mRNA via m5C.

A, I The results of RIP and RT-qPCR in H1650 and H1299 cells. IgG was used as a negative control to preclude nonspecific binding. B The results of methylated-RIP for H1650 and H1299 cells. The relative m5 C enrichment of EZH2 mRNA for each group was normalized to the Input. C EZH2 5’UTR containing either wild-type or mutant (C-to-A mutation) m5C sites was cloned into luciferase reporter vector. D Relative luciferase activity of the wild-type and mutant form of EZH2 5’UTR reporter vectors in H1299 cells transfected with sh-control or sh-NOP2, respectively. E, F The relative expression of EZH2 mRNA was detected after treating H1650 and H1299 cells with 5 μg/mL actinomycin D for indicated times. J, K Do the same. G H1299 cells in the control group and NOP2 overexpression group were treated with 100 μg/mL CHX for the indicated times, and protein expression of EZH2 was analyzed by western blot analysis. H H1299 cells transfected with sh-control or sh-NOP2 were treated with 200 ng/mL puromycin for the indicated time and the whole cell lysates were detected by western blot. L The expression level of EZH2 protein in A549 and H1299 cells of each group. M, N The results of the transwell assays. Scale bar = 100μm. Statistical methods: independent samples t-test (A, B, D, E, F, I, J, K), one-way ANOVA (N). Data are presented as the mean ± SEM of at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.