Cell cultivation and treatment

Mouse embryonic fibroblasts (MEFs) and human breast cancer cell line MCF7 were maintained in Dulbecco's modified Eagle's medium (DMEM; Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum FCS (Merck), penicillin (1 U/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere containing 5% CO2. For dependent recruitment of ac4C on cell cycle experiments, we used HeLa-Fucci cells expressing RFP-Cdt1 in the G1 phase and GFP-geminin in the S/G2/M phases, as have previously been described in detail by Sakaue-Sawano et al. [23]. HeLa-Fucci cells were cultivated in the same in vitro conditions. 53BP1-deficient and 53BP1 wild-type immortalized mouse embryonic fibroblasts (iMEFs) were a gift from Michela Di Virgilio, Laboratory of DNA Repair and Maintenance of Genome Stability, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany. Immortalized MEFs were cultured in DMEM medium supplemented with 10% FBS, 2 mM L-Glutamine, and Penicillin–Streptomycin at 37 °C and 5% CO2 [28]. RIF1-deficient HCT116 cells and wild-type HCT116 cells (a generous gift from Prof. David M. Gilbert, San Diego Biomedical Research Institute, USA, [29]) were cultivated in DMEM (Merck) supplemented with 10% FBS, and Penicillin–Streptomycin. Wild-type (wt) and NAT10 double null (dn) HeLa cells were cultured in DMEM and supplemented with 2 mM L-glutamine, 10% bovine calf serum without antibiotics, and maintained in a thermostat at 37 °C, supplemented with 5% CO2. These cells were a generous gift from Dr. Shalini Oberdoerffer, NCI NIH, USA (Arango et al. [2, 17]).

We inhibited RNA polymerase I or poly (ADP ribose) polymerase (PARP). In this case, cells were treated at 50% confluence with actinomycin D (#A9415, Merck; final concentration 0.5 µg/ml, 2 h treatment before microirradiation), or by olaparib (#S1060, Selleckchem, Germany; final concentration 10 µM, treatment 24 h before microirradiation) [30, 31].

Treatments by enzymes: We used Turbo-DNase (#AM2238, Thermo Fisher Scientific, Waltham, MA, USA), RNase A (#R5503, Merck), and RNase H1 (#EN0201, Thermo Fisher Scientific). The cells were permeabilized with cold 0.1% Triton X-100 in PBS for 10 s, washed twice in phosphate-buffered saline (PBS), and incubated in 300 μl RNase A (0,5 mg/ml in PBS) or DNase I (5 U in 1 × DNase Reaction buffer) or RNase H1 (2U in 1 × RNase Reaction buffer) for 8 min at 37 °C before immunostaining [19, 32]. Subsequently, fixation was performed with 4% formaldehyde and permeabilization with 0.3% Triton X. After that, it was followed by further enzymatic treatment for 1 h at 37 °C. Relative fluorescence intensity was evaluated in 25 nuclei, and statistical analysis was performed. For dot blot analysis, digestion was performed by RNase A (2U/5 µg RNA), RNase H1 (2U/5 µg RNA), and DNase I (0.8 U/5 µg RNA) and incubated for 15 min at 37 °C.

Irradiation by UV-light

Cells seeded on 35 mm glass-bottom dishes (#D35-20-1-N, Cellvis Mountain View, CA, USA) and at 50% confluence were sensitized with 10 μM BrdU (#11296736001, Merck) for 16 h before UVA treatment. Cells were irradiated by the UVA lamp (model GESP-15, 15 W, UVA 330–400 nm wavelength, with maximum efficiency at 365 nm) or UVC lamp (Philips, Amsterdam, The Netherlands, model TUV 30 W T8, UVC 254 nm wavelength). Irradiation was performed for 10 min. After UVC irradiation, the cells were fixed at multiple intervals (5 min, 20 min, 60 min, and 120 min after irradiation). The lamp distance from the sample was 2 cm for the UVA source and 60 cm for the UVC source [20]. Statistical analysis was performed for 40 cells.

Immunofluorescence and confocal microscopy

The immunofluorescence protocol was adapted according to Svobodova Kovaříková et al. [33] and modified. Cells were fixed with 2 ml 4% formaldehyde (prepared from paraformaldehyde, PFA; #AAJ19943K2, Thermo Fisher Scientific) for 5 min at room temperature (RT), and then 200 ml 1% SDS was added, and initial incubation was extended by an additional 7 min. Afterward, samples were permeabilized with 0.2% Triton X-100 for 15 min and washed twice in PBS for 15 min. As a blocking solution, we used 1% bovine serum albumin (Merck), dissolved in 0.1% 1 × PBS-Tween 20 (BSAT) for 1 h at RT Dishes with fixed cells were washed for 15 min in PBS and incubated with primary antibodies at a 1:100 dilution in 1% BSAT at 4 °C overnight. For immunofluorescence analysis, the following antibodies were used: anti-N4-acetylcytidine/ac4C in RNA (#A18806 Abclonal, Woburn, MA, USA), anti-phosphorylated histone H2AX (γH2AX; phospho S139) (#05-636, Merck), anti-fibrillarin (#ab4566, Abcam), anti- phospho-ATM; Ser1981 (#MAB3806-C, Merck), anti-α-tubulin (#ab80779 Abcam), and anti-NAT10 (B-4) (#sc-271770, Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with primary antibodies, the samples were washed twice in PBS for 15 min and incubated with the following secondary antibodies, diluted 1:300 in 1% BSAT: Alexa 488-conjugated goat anti-mouse (#ab150077, Abcam), Alexa 594-conjugated goat anti-rabbit (#A11037, Thermo Fisher Scientific), Alexa 488-conjugated goat anti-mouse (#A11029, Thermo Fisher Scientific), Alexa Fluor 594-conjugated goat anti-mouse (#A11032, Thermo Fisher Scientific), and Alexa 647-conjugate goat anti-rabbit (#A21245, Thermo Fisher Scientific). The DNA content was visualized using 4′,6-diamidino-2-phenylindole (DAPI; Merck), and Vectashield (Vector Laboratories, USA) was used as the mounting medium. Samples were also incubated without primary antibodies for negative control staining.

Local laser microirradiation and laser scanning confocal microscopy

For the microirradiation experiments using UVA lasers (wavelength 355 nm), cells were seeded on 35 mm gridded microscope dishes (#81,166, Ibidi, Fitchburg, WI, USA), and at 50% confluence, cells were sensitized with 10 μM BrdU for 16 h. For microscopy, the cells were maintained under optimal cultivation conditions in an incubation chamber (EMBL) at 37 °C, supplemented with 5% CO2. In the selected cell nuclei, we irradiated only the defined region of interest (ROI) using a laser connected to TCS SP5-X confocal microscope system (Leica, Wetzlar, Germany). The microscope settings for induction of local DNA damage were as follows: laser power (355 nm) 25 mW, 512 × 512 pixel resolution, 400 Hz, bidirectional mode, 48 lines, zoom 4, and 63 × oil objective (HCX PL APO, lambda blue) with a numerical aperture (NA) = 1.4 [20]. The maximum exposure of the cells to the laser was 45 min, and we monitored approximately 100 cell nuclei. Analysis of 3 biological replicates was performed. After the immunostaining procedure, locally microirradiated cells were localized according to registered coordinates on gridded microscope dishes. We studied the level of the epigenetic marker N4-acetylcytidine in RNA, NAT10 acetyltransferase, and the presence of γH2AX (phospho S139), which was also used for the optimization of microirradiation experiments. For image acquisition and analysis of fluorescence intensity (FI) we used LEICA LAS X software.

Western blotting

Western blotting was performed using the methods reported in [33]. We used the following primary antibodies: anti-phosphorylated histone H2AX (γH2AX; phospho S139; #ab2893, Abcam), anti-α-tubulin (#ab80779 Abcam), anti-NAT10 (B-4) (#sc-271770, Santa Cruz Biotechnology), and antibody against α-tubulin (#ab80779 Abcam). As secondary antibodies, we used anti-rabbit IgG (#A-4914, Merck; dilution 1:2000), anti-mouse IgG (#A-9044, Merck; dilution 1:2000) and anti-mouse IgG1 (#ab97240, Abcam; dilution 1:5000).

Isolation of total, long and small RNA

Total RNA was purified from MEFs (the second day after seeding) using the Quick-RNA Miniprep Kit (#R1054; Zymo Research, Irvine, CA, USA). For isolation of small RNA was used mirVana™ miRNA Isolation Kit (#AM1560, Thermo Fisher Scientific). RNA isolations were done according to the manufacturer's instructions. Large forms of RNA were separated using both kits in a special step according to the manufacturer's instructions. Following purification, RNA was quantified using a spectrophotometer (NanoDrop™ 2000/2000c Spectrophotometers (#ND-2000, Thermo Fisher Scientific). RNA samples for Dot blot analysis and mass spectrometry were isolated from 3 biological replicates.

Gel analysis of RNA

RNAs (1–1,5 µg) intended for dot blotting were mixed with 2 × RNA loading dye (#R0641, Thermo Fisher Scientific) in a ratio of 1:1 and heated to 70 °C for 5 min, followed by chilling on ice. These RNA samples were separated in a 10% denaturing polyacrylamide gel containing 7 M urea. As running buffer was used 1 × TBE (90 mM Tris, 90 mM borate, and 2 mM EDTA, pH = 8.0) and electrophoresis conditions were 35 mA for 1 h in the cold. After electrophoretic separation, the gel was washed in 1 × TBE for 10 min and incubated in GelRed (#41,003, Biotium, Fremont, CA, USA) for 15 min. Additional washing was in 1 × TBE. The RNA bands were visualized using an Amersham Imager 680 (GE Healthcare, Freiburg, Germany).

Dot blots

Samples of RNA were diluted to a final concentration of 250 ng/µl or 200 ng/µl. The method was based on the Abcam RNA Dot Blot Protocol (https://www.abcam.com/protocols/rna-dot-blot-protocol) and modified according to our conditions. Diluted RNA was denatured at 95 °C in a heat block for 3 min, immediately placed on ice for 1 min, and loaded onto Hybond-N + membranes. Membranes were crosslinked by UVC 254 nm lamp for 30 min. The parameters were calculated so that the total energy was 300 mJ/cm2. After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer, at 4 °C (dilution 1:2000 or 1:1000). Membranes were washed three times for 10 min in TBST. As the secondary antibody, we used anti-rabbit IgG (#A-4914, Merck; dilution 1:5000) and visualized spots by Amersham Imager 680 (GE Healthcare). The loading was determined by 0.02% methylene blue stain (R.0648.1, P-LAB, Czech Republic). Chemical deacetylation was induced by hydroxylamine (50 mM, pH = 7, 65 °C, 1 h), according to [22].

Mass spectrometry

Analysis by mass spectrometry we performed in the core facility of the Central European Institute of Technology (CEITEC) in Brno. Isolation of RNA was performed as described above, and standard N4-acetylcytidine, for mass spectrometric measurement of the ac4C level in RNA, was purchased from BIZOL company, cat. number #CBS-NA05753; CAS [3736-18-1], Biosynth; https://www.biozol.de/en/product?q=CBS-NA05753. RNA modification analysis protocol was adapted from S. Kellner et al. [34, 35]. Shortly, RNA (10 µg) was digested with 0.1U P1 nuclease, 0.3U Snake venom phosphodiesterase, and 20 ng/µl pentostatin for 2 h at 37 °C. Dephosphorylation was performed using Shrimp alkaline phosphatase in 1 × Phosphatase buffer (we added 1/10 volume of 100 mM MgCl2, 100 mM ammonium acetate, pH 9.0) for 1 h at 37 °C and filtered through Microcon-10 filters (Merck). The nucleosides were separated on a YMC-Triart C18 column (100 × 3.0 mm ID, S-3 µm, 12 nm, YMC) and analyzed using HPLC Agilent 1260 Infinity system (Agilent) (Additional file 2: Table S1). The canonical nucleoside level was measured using a 1260 infinity DAD detector and quantified using an Agilent 6460 Triple Quad Mass Spectrometer.

Statistical analysis

Fluorescence intensity values were measured by LAS X software and subsequently analyzed in Python 3 software. The obtained data were compared statistically using the ANOVA One-Way test, available in GraphPad Prism software, version 9 for Windows (GraphPad Software, San Diego, CA, USA). Also, the Student's t-test (Sigma Plot software, version 14.5; Systat Software, Inc., USA) was used for statistical analysis. All values labeled in the graphs by the asterisk(s) differ significantly from the control values.