Journal: Nature Communications

Article Title: NSUN2 facilitates DICER cleavage of DNA damage-associated R-loops to promote repair

doi: 10.1038/s41467-025-63220-9

Figure Lengend Snippet: a Left and top right: Representative proximity ligation assay (PLA) images showing the interaction between NSUN2 and γH2AX in U2OS cells treated with ionizing radiation ( + IR) or untreated (−IR) (10 Gy, 15 min). PLA signal between NSUN2 and γH2AX following IR with or without transcriptional inhibitors 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or triptolide (TPL), indicating transcription dependency. Single antibody (NSUN2 and γH2AX) and siNSUN2-treated controls confirm PLA specificity. Cells were co-stained with DAPI. Scale bars, 10 μm. Bottom right: Scattered box plot show quantification of PLA nuclear foci. **** p ≤ 0.0001. Non-significant comparisons ( p > 0.05) are not shown. b Left: representative time-lapse images of U2OS cells before and after laser-induced DNA damage. Cells were transiently transfected with either NeonGreen-tagged NSUN2 (NSUN2) or NeonGreen-only control (NeonGreen) constructs. Localised DNA double-strand breaks were generated by targeted irradiation with a 405 nm laser across a predefined region of interest (ROI), following pre-sensitisation with 10 μM Hoechst 33342 for 30 min at 37 °C to facilitate photosensitisation. Live-cell fluorescence imaging was performed over a 96-second time course. Scale bars, 5 μm. Right: quantification of fluorescence intensity as the ratio of ROI signal to background signal over time, n = 3. Data are presented as mean values ± Standard Error (SEM). Statistical analysis was performed using two-way ANOVA with multiple comparisons. **** p ≤ 0.0001. Source data are provided as Source Data file.

Article Snippet: For experiments combining PLA with γH2AX immunofluorescence, cells were incubated with anti-γH2AX antibody conjugated to FITC (Miltenyi Biotec, #130-118-339; 1:1000 dilution) for 1 h at room temperature in the dark immediately following the FISH-PLA protocol.

Techniques: Proximity Ligation Assay, Staining, Transfection, Control, Construct, Generated, Irradiation, Fluorescence, Imaging

Journal: Nature Communications

Article Title: NSUN2 facilitates DICER cleavage of DNA damage-associated R-loops to promote repair

doi: 10.1038/s41467-025-63220-9

Figure Lengend Snippet: a Left: Schematic of the FISH–proximity ligation assay (FISH-PLA) used to detect NSUN2 interactions with damage-induced antisense RNAs (DARTs) at AsiSI-induced DSBs. Upon incorporation of 5-azacytidine (zC) into nascent RNA, NSUN2 forms a covalent RNA–protein adduct. These are detected using an anti-NSUN2 antibody and a strand-specific DNA FISH probe complementary to the target RNA. Spatial proximity allows for ligation of PLA oligonucleotides, generating a red fluorescent signal. Created in BioRender. Gullerova, M. ( https://BioRender.com/ ) ys14435. Right: Design of strand-specific FISH probe sets targeting either antisense (DS2-AS, blue) or sense (DS2-SS, orange) DARTs at the DS2 AsiSI cut site. Each probe set includes ten oligonucleotides spaced ~40 bp apart, starting ~100 bp from the DSB. The genomic context of the DS2 site is shown with annotated VSTM2B , an AsiSI target gene, and RNA Pol II directionality, enabling strand-resolved detection of damage-induced RNA–protein complexes. b Representative FISH-PLA images showing NSUN2–RNA adduct detection at the DS2 antisense strand in DIvA U2OS cells. Cells were pre-treated with zC (1 h, 37 °C) to trap NSUN2, followed by DSB induction with (Z)−4-hydroxytamoxifen (+4OHT). PLA was performed using probes against NSUN2 and antisense DARTs. Red puncta indicate NSUN2–RNA proximity signals; nuclei were stained with DAPI (blue). Shown are untreated ( − 4OHT) and damaged ( + 4OHT) conditions, along with controls: transcription inhibition (triptolide, TPL), RNase H digestion of R-loops, and Exonuclease VII treatment to degrade 3′ ssDNA. All treatments were applied post-fixation. Images were acquired under identical settings. Scale bars, 10 μm. c Scattered box plot quantifying zC-FISH-PLA foci per nucleus. Statistical significance: * p ≤ 0.05; **** p ≤ 0.0001; ns ( p > 0.05) not shown. d Representative single-plane images showing zC-FISH-PLA detection of NSUN2 and DS2 antisense RNA (red), with immunofluorescence staining for γH2AX (green). Nuclei are counterstained with DAPI (blue). Merged images show co-localisation of NSUN2–RNA complexes with DNA damage foci. White boxes indicate regions magnified in lower panels. Scale bars: overview, 10 μm; zoomed insets, 3 μm. n = 43 cells analysed. Source data are provided as Source Data file.

Article Snippet: For experiments combining PLA with γH2AX immunofluorescence, cells were incubated with anti-γH2AX antibody conjugated to FITC (Miltenyi Biotec, #130-118-339; 1:1000 dilution) for 1 h at room temperature in the dark immediately following the FISH-PLA protocol.

Techniques: Proximity Ligation Assay, Ligation, Staining, Inhibition, Immunofluorescence

Journal: Nature Communications

Article Title: NSUN2 facilitates DICER cleavage of DNA damage-associated R-loops to promote repair

doi: 10.1038/s41467-025-63220-9

Figure Lengend Snippet: a Surface plasmon resonance (SPR) sensorgrams (left) showing real-time binding responses of recombinant DICER (0.00117–0.6 µM) to immobilized NSUN2. Response units (RU) are plotted against time to show association and dissociation phases. Right: equilibrium binding curve plotting steady-state RU values against DICER concentration. Fitted data yield an estimated dissociation constant (K_d ≈ 0.5 µM). b Left: representative proximity ligation assay (PLA) images detecting NSUN2–DICER interaction in U2OS cells following DNA damage induced by ionizing radiation ( + IR) or (Z)−4-hydroxytamoxifen (+4OHT). A single-antibody control (anti-DICER) confirms signal specificity. Scale bars, 10 μm. Right: Scattered box plot quantifying PLA signals across conditions. Treatments include transcription inhibition (DRB, triptolide), and post-fixation RNase digestion (RNase A, T1, III) to assess RNA dependence. Significance: *** p ≤ 0.001; **** p ≤ 0.0001; ns ( p > 0.05) not shown. c Left: representative images showing FISH-PLA detection of DICER proximity to DS2 antisense RNA in DIvA U2OS cells after +4OHT-induced DSBs. Scale bars, 10 μm. Right: Quantification of PLA foci per nucleus under multiple conditions: untreated or +4OHT-treated cells, NSUN2 or DICER knockdown (siNSUN2, siDICER), scrambled probe control (SCR DS2), and RNase H treatment to degrade R-loops. Significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns not shown. d Metagene plots showing ChIP enrichment for NSUN2 (top) and DICER (bottom) in AsiSI-ER U2OS cells treated with +4OHT. Signals are plotted as normalized read coverage across ±2.5 kb surrounding 80 AsiSI-induced DSBs (BLESS80), centred at the cut site (0). NSUN2 signal (red) is compared to Input (blue); DICER signal is shown in green. e Single-plane images showing FISH-PLA signal (red) for DICER–DS2 antisense RNA complexes co-stained with γH2AX (green) after +4OHT treatment. Merged images highlight co-localisation at DNA damage foci. White boxes mark zoomed-in regions (right panels). Scale bars: overview, 10 μm; zoom, 3 μm. n = 52 cells analysed. Source data are provided as Source Data file.

Article Snippet: For experiments combining PLA with γH2AX immunofluorescence, cells were incubated with anti-γH2AX antibody conjugated to FITC (Miltenyi Biotec, #130-118-339; 1:1000 dilution) for 1 h at room temperature in the dark immediately following the FISH-PLA protocol.

Techniques: SPR Assay, Binding Assay, Recombinant, Concentration Assay, Proximity Ligation Assay, Control, Inhibition, Knockdown, Staining

Journal: Nature Communications

Article Title: NSUN2 facilitates DICER cleavage of DNA damage-associated R-loops to promote repair

doi: 10.1038/s41467-025-63220-9

Figure Lengend Snippet: a Left: Representative Comet assay images of U2OS cells treated with ionizing radiation (IR, 5 Gy, 15 min) and harvested at 1 h (+IR 1 h) or 24 h (+IR 24 h), or left untreated (−IR). Cells were transfected with siRNAs targeting NSUN2 (siNSUN2), RAD51 (siRAD51), or a negative control (siNC). Right: Quantification of DNA damage by tail moment. Data represent mean ± SD (n = 3). Statistical analysis was performed using a two-tailed Mann–Whitney test. Significance: **** p ≤ 0.0001; ns (p > 0.05) not shown. b MTS assay measuring cell viability in HeLa cells transfected with siNSUN2, siRAD51, or siNC, and irradiated with 5 Gy. Cell viability was assessed at 24 h, 48 h, and 72 h post-irradiation. Data are shown as mean ± SD ( n = 5). Statistical significance was determined by two-tailed Mann–Whitney test. ** p ≤ 0.01; *** p ≤ 0.001; ns not shown. c Schematic of the DR-GFP reporter system used to assess homologous recombination (HR) repair efficiency. Quantification of HR activity is shown in U2OS cells transfected with siNSUN2, siRAD51, both siRNAs (siRAD51 + siNSUN2), or siNC. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns not shown. d Left: Immunofluorescence images showing γH2AX foci formation in U2OS cells under the following conditions: siNC, siNSUN2, NSUN2 knockdown rescued with FLAG-tagged NSUN2 wild-type (siNSUN2 + NSUN2 wt) or methyltransferase-dead mutant (siNSUN2 + NSUN2 K190M). Cells were irradiated with 5 Gy and fixed at 4 h post-IR. γH2AX staining (green) marks DNA damage; FLAG-NSUN2 variants were used to assess rescue efficiency. Scale bars, 10 μm. Right: Quantification of γH2AX signal intensity per nucleus. Significance: **** p ≤ 0.0001; ns not shown. Source data are provided as Source Data file.

Article Snippet: For experiments combining PLA with γH2AX immunofluorescence, cells were incubated with anti-γH2AX antibody conjugated to FITC (Miltenyi Biotec, #130-118-339; 1:1000 dilution) for 1 h at room temperature in the dark immediately following the FISH-PLA protocol.

Techniques: Single Cell Gel Electrophoresis, Transfection, Negative Control, Two Tailed Test, MANN-WHITNEY, MTS Assay, Irradiation, Homologous Recombination, Activity Assay, Immunofluorescence, Knockdown, Mutagenesis, Staining