Fig. 3
Recruitment of ac4C RNAs to UVA- and UVC-damaged chromatin. A Local microirradiation by 355-nm laser line showed that ac4C RNA recognizes UVA-microirradiated chromatin immediately after laser irradiation. In the later stages of DDR, 11–45 min post-irradiation, the ac4C RNA signal at DNA lesions was reduced. Scale bars are 5 µm. B, C Dot blot analysis of ac4C RNA documents levels of ac4C in total RNA isolated from non-irradiated, UVA-, and UVC-irradiated MEF cells. B shows the level of ac4C in RNAs (#ab252215, Abcam) studied 5 min and 30 min after UVC irradiation, and panel C documents the density of ac4C in RNAs (#ab252215, Abcam) analyzed 15 min after UVA and UVC irradiation. Negative controls (samples not incubated with the primary antibody) are shown. After fractionalization, it was observed that both large RNAs and small RNAs were notably acetylated on N4-cytidine when the cells were exposed to UVC light for 5 min, while 15 min post-irradiation, the highest level of ac4C was on small RNAs. Quantification of the density of dot spots is shown for both panels B and C. D Representative anti-ac4C dot blot (#ab252215, Abcam) performed on N4-acetylcytidine (NA05753, Biosynth). Chemical deacetylation was induced by hydroxylamine (50 mM, pH7, 65 °C, 1 h) [22]. The specificity of N4-acetylcytidine (NA05753, Biosynth) was verified by antibody against m6A in RNA (#202 111, SYSY Antibodies). E Mass spectrometry data on ac4C in total and large RNAs studied in control, non-treated cells, and cells exposed to UVA, UVC light, and γ-rays. Cells were also treated with actinomycin D (ActD). E shows the mean ± standard deviations (SD) for three biological replicates (n = 3). Asterisks (*) show p ≤ 0.05, calculated using the two-tailed Student's t-test, indicating statistically significant differences in the level of ac4C RNA