Figure 6

Site-specific RNA N6-methyladenosine demethylation by dCas9-FTO. (a) The dCas9–FTO fusion protein is guided to the respective target RNA via sgRNA and PAMmer to recruit it close to the m6A site for targeted demethylation. The figure on the right shows the methylation sites being targeted. (b) Confirmation that dCas9–FTO acts in the cytoplasm. When sgRNA and PAMmer were transfected, dCas9–FTO, which was present in the nucleus, was also transferred to the cytoplasm. This indicates that dCas9 acts on the target mRNA in the cytoplasm. Uncropped images are shown in Supplementary Fig. 7. (c) MeRIP–qPCR analysis shows that in the dCas9–FTO system, co-transfection of sgRNA and PAMmer reduces methylation of the PLK1 3′UTR. The degree of methylation is indicated by the %input in the vehicle transfection of each cell line (vehicle, dCas9 only, and dCas9–FTO, respectively). Values are the mean ± standard deviation of 3 and the p values in *p < 0.01 and **p < 0.05 were determined by two-tailed paired-samples t-tests. (d) Cell cycle changes induced by PLK1 3′UTR demethylation. Co-transfection of sgRNA and PAMmer increased not only the S–G2–M phase but also the sub-G1 peak. (e) Annexin V staining assay indicates that PLK1 3′UTR demethylation increased Annexin V- and 7-AAD-positive populations, which are at the terminal stage of cell death. (f) Altered expression of PLK1 by PLK1 3′UTR demethylation. No significant variation in PLK1 expression in adherent cells was observed 48 h after the transfection; however, PLK1 expression was significantly decreased in floating cells (left: mRNA, right: protein). Uncropped images are shown in Supplementary Fig. 7.