PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks

Abstract

After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.

Figure 1.

NONO increases cell survival after ionizing irradiation. (A) Western blot analysis demonstrating the efficiency of siRNAs directed against NONO (lane 2) or SFPQ (lane 3) in HeLa cells. (B) The clonogenic survival of HeLa cells treated with a scrambled control siRNA (image 1) and a siRNA directed against NONO (image 2) was analysed using a colony forming assay. (C) Quantitation of cell survival. HeLa cell colonies were counted 10 days after γ-irradiation with 0, 0.5 and 2.0 Gy.

Figure 2.

Attenuation of NONO decelerates NHEJ. (A) HeLa cells knocked-down with a scrambled siRNA (lanes 3 and 4) or a siRNA directed against NONO (lanes 5 and 6) were treated for 2 hr with NCS (500 ng/ml) and allowed to recover for either 60 or 120 min. Cells were then collected, embedded and lysed in agarose blocks and used for pulse-field gel electrophoresis. (B) A linearized, ³²P-end-labeled pBluescript was incubated for the indicated times with a nuclear extract derived from HeLa cells treated with a scrambled siRNA (lanes 2–9) or an siRNA directed against NONO (lanes 10–17). (C) Quantitation of the end joining events using a phosphorimager: The percent end joining represents the total signal intensity per lane normalized to 100% from which is substracted the % intensity of the remaining template (n = 4).

Figure 3.

NONO is a chromatin-associated protein and localizes to a unique DSB in vivo. (A) Unfixed HeLa cells were treated for 1 hr with 10 µM ABT-888, washed with PBS and fractionated into chromatin-enriched, nuclear soluble and cytoplasmic fractions. Fractions were used for an analysis by western blotting. (B) Chromatin immunoprecipitation of NONO from the fixed chromatin of MCF-7 cells, which priorly had been transfected with an I-SceI coding plasmid to generate a unique DSB. An IgG antibody served as a control for the ChIP-experiment. (C) Distribution of primer pairs relative to the DSB created by I-SceI. These primers were used in q-PCR analysis of ChIP shown in (D). Primers for GAPDH served as a control for the PCR. (D) Quantification of NONO relative to the DSB by PCR (n = 3).

Figure 4.

NONO binds PAR in vitro. (A) SDS-PAGE of 100 ng purified His-NONO protein stained with Coomassie blue (lane 2). (B) In vitro PAR-binding assay. 1 µg of purified His-NONO was loaded on an SDS-PAGE, blotted onto a nitrocellulose membrane and incubated in 250 nM ³²P-labeled PAR in TBS-T without (lane 1), with 1-fold (lane 2), 10-fold (lane 3) or 100-fold unlabeled competitor PAR (lane 4). (C) A PAR-binding assay was conducted as in (B) without (lane 1), with 1-fold (lane 2), 10-fold (lane 3) or 100-fold unlabeled competitor RNA (lane 4). (D) Kinetics of PAR binding to purified His-tagged NONO conducted by SPR spectroscopy. To analyse binding kinetics, PAR was injected at three different concentrations (125, 250 and 500 nM). PAR injection was done for 300 s and dissociation data were collected for 600 s. Data were fitted with Langmuir 1:1 interaction plot to calculate rate constants. The sensorgram is representative of three independent experiments.

Figure 5.

NONO binds PAR through its N-terminal/RNA recognition motif 1 (RRM1). (A) Protein truncations of NONO flanking the protein domains of interest, namely RRM1 and RRM2. (B) SYPRO protein stain of protein fragments loaded on a SDS-PAGE. (C) In vitro PAR-binding assay using 250 nM ³²P-labeled PAR in TBS-T. (D) SDS-PAGE of 500 ng His-NONO (lane 2) and His-NONOΔRRM1 (lane 3) each. (E) 1 µg of NONO-WT and NONOΔRRM1 purified proteins were slot blotted onto a nitrocellulose membrane and an in vitro ³²P-labeled PAR-binding assay was conducted in TBS-T. Mean values of the radioactivity signal as quantified by a phosphorimager from three independent experiments are presented.

Figure 6.

NONO is recruited to DNA damage sites in a PAR-dependent manner. (A) Representative images of laser-irradiated HeLa cells expressing GFP-NONO and subjected to IF for detection of PARP-1 and PAR. (B) Representative images of the laser-irradiated cells. HeLa cells were transfected either with the GFP-tagged NONO construct or with a mutant lacking the RRM1. Then cells were either left untreated, treated with 10 µM ABT-888 1 hr before irradiation or cotransfected with mCherry-PARG prior to laser microirradiation. (C) Statistical analysis of the recruitment kinetics. At least 15 cells per condition in three independent experiments were analysed for their fluorescence intensity above the background.

Figure 7.

Representative images of laser-irradiated MEFs that were either proficient for PARP-1 and PARP-2 (MEF-WT) or deficient for either PARP-1 (PARP-1^−/−) or PARP-2 (PARP-2^−/−). Cells had been transfected with a GFP-NONO construct 24 hr before laser micro-irradiation. At least 20 cells per condition were tested in two independent experiments. Recruitment has been observed in none of the PARP-1^−/− MEFs.

Figure 8.

Attenuation of NONO decreases NHEJ and increases HR. (A) Schematic representation of the I-SceI-based NHEJ in vivo reporter system. (B) Schematic representation of the I-SceI-based HR in vivo reporter system. (C) NHEJ repair rates in percent with siCTRL or siNONO and with or without 10 μM of the PARP-inhibitor ABT-888. The siCTRL condition was normalized to 100% (n = 3). (D) Diagram of the HR repair rates after treatment with siCTRL or siNONO and with or without 10 μM ABT-888. The siCTRL condition was normalized to 100% (n = 3).