Fig. 1: m⁵C mRNA methylation is enriched at transcriptionally active sites with DNA damage.

a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m⁵C foci from three independent experiments, mean ± SD (upper right). Fold increase of m⁵C mean intensity = mean intensity of m⁵C at TA-KR/mean intensity of background (n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m⁵C with four different anti-m⁵C antibodies. Frequency of m⁵C-positive cells in 500 cells was quantified (n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m⁵C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H₂O₂ for 40 min was used for m⁵C measurement via dot blot. Quantification of m⁵C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e ³²P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m⁵C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m⁵C are labeled (Left). f ³²P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H₂O₂ for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m⁵C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.