Figure 2.

Methylation of circRNA affects translation efficiency. (A) m⁶A in circRNA is reduced by FTO. FTO expression vector was co-transfected with circRNA containing RSV or RSV-mut m⁶A site into 293 cells, and the RNAs from transfected cells were pulled down by m⁶A-specific antibody and analyzed by RT-qPCR. The SON mRNA known to contain multiple m⁶A sites and GAPDH mRNA containing no m⁶A modification were used as controls. Control antibody is anti-GAPDH antibody. The IP experiments were repeated three times, with mean and SD plotted. (B) FTO reduces circRNA translation. RNA and protein were analyzed by semi-quantitative RT-PCR and western blots using 293 cells transfected with circRNA reporter containing RSV and FTO (or mock control). (C) METTL3 and METTL14 can methylate circRNA. circRNA with RSV or RSV-mut, METTL3 and METTL14 overexpression plasmids were co-transfected into 293 cells as in A (n = 3; mean ± SD). (D) circRNA translation is increased by METTL3/14. Experimental procedures are the same as in B. (E) 293 cells transiently expressing circRNA with RSV were subjected to heat shock stress. Cells were collected at 0, 1, 2, 4 h after heat shock (1 h at 42 °C) to analyze RNA and protein expression using semi-quantitative RT-PCR and western blots. N, no heat shock. (F) Quantification of circRNA RNA and GFP protein levels in heat-shocked cells. GAPDH levels were used for normalization (n = 3, mean ± SD).