LncRNA CTBP1-DT-encoded microprotein DDUP sustains DNA damage response signalling to trigger dual DNA repair mechanisms

Abstract

Sustaining DNA damage response (DDR) signalling via retention of DDR factors at damaged sites is important for transmitting damage-sensing and repair signals. Herein, we found that DNA damage provoked the association of ribosomes with IRES region in lncRNA CTBP1-DT, which overcame the negative effect of upstream open reading frames (uORFs), and elicited the novel microprotein DNA damage-upregulated protein (DDUP) translation via a cap-independent translation mechanism. Activated ATR kinase-mediated phosphorylation of DDUP induced a drastic 'dense-to-loose' conformational change, which sustained the RAD18/RAD51C and RAD18/PCNA complex at damaged sites and initiated RAD51C-mediated homologous recombination and PCNA-mediated post-replication repair mechanisms. Importantly, treatment with ATR inhibitor abolished the effect of DDUP on chromatin retention of RAD51C and PCNA, thereby leading to hypersensitivity of cancer cells to DNA-damaging chemotherapeutics. Taken together, our results uncover a plausible mechanism underlying the DDR sustaining and might represent an attractive therapeutic strategy in improvement of DNA damage-based anticancer therapies.

Figure 1.

LncRNA CTBP1-DT-encoded microprotein DDUP promotes DNA damage repair. (A) Left and middle panel: polysome profiling was performed to identify potential new factors involved in DNA damage responses. DNA damage in 293T cells was determined from γ-H2AX expression after CPT (10 μM) treatment for 1 h. Right: volcano plot analysis of dysregulated ribosome-associated RNAs from polysome profiling data; x-axis = log₂ fold change in expression of polysome-associated RNAs between CPT- and vehicle-treated cells; y-axis = FDR value (–log₁₀ transformed) of polysome-associated RNAs. (B) Potential ORF of the DDUP protein located in exon 2 of non-coding lncRNA CTBP1-DT in the human genome, and the full-length DDUP protein synthesised to generate a polyclone antibody. Molecular weight (MW):19.74 kDa. (C) Unique CTBP1-DT peptide identified by LC–MS/MS analysis. (D) IB analysis of DDUP expression in Flag-tagged DDUP-transduced 293T and HeLa cells validated by the generated anti-DDUP polyantibody. Synthesised DDUP protein served as a positive control and GAPDH served as a loading control. (E) IB analysis of endogenous DDUP expression in parental and CTBP1-DT-silenced 293T cells treated with CPT (10 μM) at the indicated time-point. GAPDH served as a loading control. F. IB analysis of endogenous DDUP and γ-H2AX expression in cells treated with CPT (10 μM) or VP-16 (10 μM) or CDDP (5 μM) for 1 h or with IR (10 Gy) and were allowed to recover for 6 h. GAPDH served as a loading control. (G) Real-time analysis of polysome-associated CTBP1-DT levels in CPT (10 μM)-treated cells at the indicated time-point. (H) Representative images (left) and quantification (right) of damaged DNA in the indicated cells analysed by comet assay (n = 100). The indicated cells were treated with CPT (10 μM) or VP-16 (10 μM) for 1 h. Scale bar = 20 μm. (I) Representative images (left) and quantification (right) of γ-H2AX foci in the indicated cells with CPT treatment (10 μM, 1 h). At least 100 cells were counted. Scale bar = 5 μm. (J) Representative images (left) and kinetics (right) of γ-H2AX signals in response to laser micro-irradiation in the indicated cells and recovery for the indicated times (n = 100). Each error bar represents the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2.

DDUP microprotein, but not CTBP1-DT RNA, promotes DNA damage repair. (A, B) Diagram (A) and expression (B) of the indicated lncRNA CTBP1-DT constructs, including wild-type (WT), ATG1 mutation, ATG2 mutation and double ATG site mutation (ATG mutated to ATT) in CPT (10 μM, 1 h)-treated 293T cells. GAPDH served as a loading control. (C) Representative images (left) and quantification (right) of damaged DNA in the indicated cells analysed by comet assay (n = 100). The indicated cells were treated with CPT (10 μM) for 1 h. Scale bar = 20 μm. (D) Representative images (left) and quantification (right) of γ-H2AX foci in the indicated cells with CPT treatment (10 μM, 1 h). At least 100 cells were counted. Scale bar = 5 μm. (E) Representative images (left) and kinetics (right) of γ-H2AX signals in response to laser micro-irradiation in the indicated cells and recovery for the indicated times (n = 100). (F) IB analysis of endogenous DDUP expression in CPT (10 μM, 1 h)-treated control and DDUP-KO cells. GAPDH served as a loading control. (G) Representative images (left) and quantification (right) of γ-H2AX foci in the indicated cells with CPT treatment (10 μM, 1 h). At least 100 cells were counted. Scale bar = 20 μm. (H) Representative images (left) and quantification (right) of damaged DNA in the indicated cells analysed by comet assay (n = 100). The indicated cells were treated with CPT (10 μM) for 1 h. Scale bar = 5 μm. (I) Representative images (upper) and kinetics (lower) of γ-H2AX signals in response to laser micro-irradiation in the indicated cells and recovery for the indicated times (n = 100). Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 3.

The IRES in the 5′UTR of CTBP1-DT is essential for DNA damage-induced DDUP translation. (A) IB analysis of DDUP expression in 293T and HeLa cells treated with CPT (10 μM), CPT (10 μM) + CHX (5 μg/ml), or CPT (10 μM) + ACTD (5 μg/ml) for the indicated times. GAPDH served as a loading control. (B) IB analysis of DDUP expression in 293T and HeLa cells treated with CPT (10 μM), CPT (10 μM) + 4EGI-1 (25 μM) or CPT (10 μM) + rapamycin (100 nM) for the indicated times. GAPDH served as a loading control. (C) In vitro translation assay analysis of DDUP protein in vehicle- or CPT (10 μM, 1 h)-treated DDUP-KO cell lysates using in vitro-transcribed m7G-capped, non-functional ApppG capped-, or non-capped-CTBP1-DT RNA as template. GAPDH served as a loading control for the levels of cell lysates used for in vitro translation assays. (D) Upper: Graphical representation (upper) of the location of the uORFs, IRES and DDUP ORF, and the 5′UTR and 3′UTR in CTBP1-DT. Lower: expression of DDUP in the vehicle- and CPT (10 μM, 1 h)-treated DDUP-KO cells transfected with the indicated constructs. GAPDH served as a loading control. (E) Luciferase reporter assay analysis of relative Luc/Rluc activity in vehicle- and CPT (10 μM, 1 h)-treated 293T cells transfected with the indicated constructs. (F) Representative fluorescence images of mCherry and GFP signals in vehicle- and CPT (10 μM, 1 h)-treated 293T cells transfected with the indicated constructs. Scale bar = 50 μm. (G) Representative images (left) and kinetics (right) of γ-H2AX signals in response to laser micro-irradiation in the indicated cells and recovery for the indicated times (n = 100). Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 4.

Phosphorylation of DDUP is essential for DDUP-mediated damage repair. (A) IF staining analysis of endogenous DDUP foci using anti-DDUP or Flag-tagged DDUP foci using anti-Flag antibody in vector- or Flag-tagged DDUP-transfected HeLa cells treated with vehicle, CPT (10 μM) or CDDP (5 μM) for 1 h. (B) Chromatin fraction and IB analysis of DNA-bound DDUP in vector- and Flag-tagged DDUP-transfected HeLa cells treated with vehicle, CPT (10 μM) or CDDP (5 μM) for 1 h. H3 served as a loading control. (C) LFQ analysis of potential significantly upregulated DDUP-interacting proteins in vehicle- and CPT (10 μM, 1 h)-treated 293T cells. (D) Co-IP analysis of the interaction of DDUP with ATR, ATM,RAD18, γ-H2AX, RAD51C, p-CHK1, CHK1 and PARP1 in CPT (10 μM, 1 h)—and CDDP (5 μM, 1 h)-treated 293T cells with or without berzosertib (80 nM, 1 h) treatment. (E) IF staining analysis of DNA damage-induced p-ATR foci (red) and endogenous DDUP foci (green) in HeLa cells treated with CPT (10 μM), CDDP (5 μM), or combination with berzosertib (80 nM) for 1 h. (F) Far-western blotting analysis of the direct ATR/DDUP interaction using anti-ATR antibody-immunoprecipitated proteins and detected using anti-DDUP antibody then re-blotting with anti-ATR antibody. Recombinant DDUP protein served as a control. (G) Molecular docking between ATR and DDUP performed using the Cluspro 2.0 web server (https://cluspro.org/help.php). The structure is shown in cartoon representation. The 3D structure of WT DDUP was obtained from the I-TASSER server and the 3D structure of ATR (PDB ID: 5yz0) was downloaded from the RCSB Protein Data Bank. (H) Schematic illustration of full-length and truncated DDUP proteins (upper) and co-IP assay analysis of the ATR-interacting region in DDUP using anti-ATR antibody in for CPT (10 μM, 1 h)-treated HeLa cells transfected with full-length and truncated DDUP fragments (lower). (I) IP assays using anti-Flag antibody performed in DDUP/WT- and DDUP/T174A mutant-transfected cells treated with ATR inhibitor (80 nM), ATM inhibitor (10 μM), or DNA-PKcs inhibitor (2 μM) for 1 h prior to treat with or without CPT (10 μM, 1 h) as indicated, analysed by immunoblotting with anti-pTQ/SQ antibody. (J) IF staining using anti-DDUP antibody performed in DDUP/WT- and DDUP/mutant-transfected cells with or without CPT treatment (10 μM, 1 h), with the image captured by laser confocal microscopy. Scale bar = 5 μm. (K) Chromatin fraction and IB analysis of DNA-bound DDUP/WT, DDUP/T174A and DDUP/T174D in CPT (10 μM, 1 h)-, CDDP (5 μM, 1 h)- and IR (10 Gy)-treated DDUP-KO HeLa cells transfected with DDUP/WT and DDUP/mutant plasmids. (L) Representative images (left) and quantification (right) of γ-H2AX foci in the CPT (10 μM, 1 h)-treated indicated cells with or without berzosertib treatment (80 nM, 1 h). At least 100 cells were counted. Scale bar = 5 μm. (M) Kinetics of γ-H2AX signals in response to laser micro-irradiation in the indicated cells and recovery for the indicated times (n = 100). The indicated cells treated with or without berzosertib (80 nM) for 1 h. Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 5.

Phosphorylated DDUP forms a complex with γ-H2AX and RAD18. (A) IF staining analysis of the co-localisation of DDUP foci with RAD18 foci, γ-H2AX foci, RAD51C-foci and PARP1-foci in vehicle- and CPT (10 μM, 1 h)-treated HeLa cells. Scale bar = 5 μm. Co-localization the fluorescence between molecules was quantified using the Manders' overlap coefficients algorithm. (B) Co-IP assay analysis of the formation of the DDUP/γ-H2AX/RAD18/RAD51C complex in the indicated gene-silenced 293T cells treated with CPT (10 μM) for 1 h. (C) Co-IP assay analysis of the interaction of WT and mutated DDUP with γ-H2AX, RAD18 and RAD51C in CPT (10 μM, 1 h) or without CPT-treated 293T cells. (D) Far-western blotting analysis of the direct interaction of DDUP/γ-H2AX using anti-γ-H2AX antibody-immunoprecipitated proteins (left), or DDUP/RAD18 using anti-RAD18 antibody-immunoprecipitated proteins (middle), or DDUP/RAD51C using anti-RAD51C antibody-immunoprecipitated proteins in RAD18-silenced cells (right), then detected using anti-DDUP antibody. Recombinant DDUP/T174D protein served as control. (E) Left: the 3D structure of WT DDUP in the dense state obtained from the I-TASSER server. Right: the 3D structure of the DDUP mutant (T174 to D174), which mimics phosphorylation of DDUP, in the loose state obtained from the I-TASSER server. (F) Molecular docking of H2A.X and DDUP. The 3D structure of H2A.X obtained from the I-TASSER server. The combined surface, cartoon and stick representation shows the predicted interaction interface between H2A.X and DDUP based on the modeled DDUP structure. The DDUP protein is coloured pink and H2A.X is coloured in cyan. (G) Molecular docking of RAD18 and DDUP. The 3D structure of RAD18 (PDB ID: 2YBF) was download from the RCSB Protein Data Bank. The combined surface, cartoon and stick representation shows the predicted interaction interface between RAD18 and DDUP based on the modeled DDUP structure. The DDUP protein is coloured pink and RAD18 is coloured cyan. (H) Upper: Schematic illustration of WT and truncated DDUP proteins. Lower: IP assay analysis of the γ-H2AX-interacting region of DDUP using anti-γ-H2AX antibody (lower left) and the RAD18-interacting region of DDUP using anti-RAD18 antibody in CPT (10 μM, 1 h)-treated 293T cells transfected with full-length and truncated DDUP fragments. (I) SPR analysis of the direct interaction between DDUP and γ-H2AX (left) and the direct interaction between DDUP and RAD18 (right). DDUP protein was immobilised on a Series S Sensor Chip. The K_d value for the DDUP/γ-H2AX and DDUP/RAD18 interaction was calculated as the raw response (RU). Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 6.

DDUP enhances the retention of RAD18 at DNA damage sites. (A) Representative images (left) and time course (right) of the formation of CPT (10 μM)-induced RAD18 and RAD51C foci in control and DDUP-KO HeLa cells and allowed to recover for the indicated times. The RAD18- and RAD51C foci was examined every 10 min in the CPT-treated cells within the first 2 h. Cells containing more than 10 RAD18 and RAD51C foci per nucleus were scored. (B) Chromatin fraction and IB analysis of DNA-bound RAD18, RAD51C and DDUP in the indicated CPT (10 μM)-treated cells and allowed to recover for the indicated times. H3 served as a loading control. (C) Quantitative FRAP analysis of GFP-RAD18 in GFP-RAD18-transfected control and DDUP-KO HeLa cells (right), and in DDUP-KO HeLa cells co-transfected with GFP-RAD18 and vector, GFP-RAD18, and DDUP/WT, or GFP-RAD18 and DDUP/T174A, treated with CPT (10 μM) and allowed to recover for the indicated times. (D) Kinetics of γ-H2AX signals in the indicated cells in response to laser micro-irradiation and allowed to recover for the indicated times (n = 100). (E) IP assay analysis of the DDUP/RAD51C and DDUP/PCNA interaction in control and RAD18-silenced 293T cells treated with CDDP (5 μM, 1 h). (F) Homologous recombination repair assays performed in the indicated cells. (G) IP/IB analysis of the regulatory effect of DDUP dysregulation on PCNA monoubiquitination in the indicated cells treated with CDDP (5 μM, 1 h) or UV radiation (60 J/m²). H3 and α-tubulin served as loading control. Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Figure 7.

Upregulation of DDUP confers resistance to cisplatin in ovarian cancer cells in vitro. (A) Representative images of DDUP expression in chemo-sensitive and chemo-resistant ovarian cancer cells (n = 367) (left) and the positive correlation between DDUP levels and platinum resistance (P < 0.001; r = 0.502) and relapse (P < 0.001; r = 0.389) in ovarian cancer tissues (n = 367; right). Scale bar = 20 μm. Chi-square test was used for statistical analysis. (B) Kaplan–Meier analysis of overall survival (left) and relapse-free survival (right) for patients with ovarian cancer stratified by low versus high expression of DDUP (log-rank test; p < 0.001; n = 367). (C) IB analysis of expression of DNA-bound and total DDUP in patient-derived ovarian cancer cells (PDOVCs). H3 and GAPDH served as a loading control. (D) Homologous recombination repair assays performed in PDOVCs transfected with DR-GFP and pCBAScel plasmids following treatment with CDDP (5 μM) alone or CDDP (5 μM) plus Berzosertib (80 nM) for 1 h. (E) IB analysis of expression of DNA-bound and total monoubiquitinated PCNA and DDUP in the indicated PDOVCs treated with CDDP (5 μM) alone or CDDP (5 μM) plus Berzosertib (80 nM) for 1 h. H3 and α-Tubulin served as loading control. (F) Representative images (left) and quantification (right) of γ-H2AX foci determined by IF staining using anti-γ-H2AX antibody and damaged DNA determined by comet assay in the indicated PDOVCs treated with CDDP (5 μM, 1 h). Scale bar = 5 μm (left) and 20 μm (right). (G) Representative images (upper) and quantification (lower) of the number of PDOVCs colonies following the indicated treatment. Each error bar represents the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 8.

Hypothetical model. DDUP encoded by an lncRNA orchestrates DNA damage repair by regulating PCNA monoubiquitination and RAD18 retention at the DNA damage sites.