Figure 3.

NSun2 methylates ATX mRNA mainly at C2756.A, brief pattern diagram of bisulfite RNA-Seq method. B, in vitro transcribed ATX mRNA 3′UTR-A fragment was methylated by NSun2, and then bisulfite RNA-Seq analysis was conducted to identify the methylation site. The percentages of different m⁵C methylation sites in ATX mRNA 3′UTR-A were indicated. C, ATX mRNA 3′UTR, 3′UTR-A, 3′UTR-M (cytosine 2756 mutated to thymidine in 3′UTR), and 3′UTR-A-M (cytosine 2756 mutated to thymidine in 3′UTR-A) fragments were used to perform the in vitro methylation analysis. The incorporation of ³H-labeled SAM into each fragment was measured by liquid scintillation counting. ATX cDNA and bacterial tRNA served as the negative control and positive control, respectively. D, U87 cells were transfected with NC siRNA or NSun2 siRNA, and total RNA was isolated at 48 h after transfection. RNA was subjected to bisulfite sequencing analysis, and the levels of C2756 and C2757 methylation in ATX mRNA were assessed. Open boxes at positions of C2756 and C2757 indicate the unmethylated cytosines, which were converted to uracil and read as thymidine in ATX cDNA. Filled boxes at C2756 and C2757 indicate the methylated cytosines, which retained as cytosine in ATX cDNA. The numbers showed the residue positions in ATX mRNA with the first residue of 5′UTR as first. All the data are presented as the mean ± S.E. of the results of three independent experiments. p values were calculated by two-sided unpaired Student's t test; ns, not significant; ***p < 0.001.