Ribonucleotide incorporation into mitochondrial DNA drives inflammation

Cell culture, transfection and RNA interference

MEFs and HeLa cells were maintained in DMEM-GlutaMAX (Thermo Fisher Scientific) containing 4.5 g l⁻¹ of glucose supplemented with 1 mM sodium pyruvate (Gibco), 100 μM non-essential amino acids (Gibco) and 10% FBS (Biochrom). IMR90 cells were maintained in MEM-GlutaMAX (Thermo Fisher Scientific) and 10% FBS. MEF and HeLa cell lines were maintained at 37 °C under 5% CO₂ and ambient oxygen. IMR90 cells were maintained at 37 °C under 5% CO₂ and 3% O₂. Cell lines were routinely tested for Mycoplasma infection. Cell numbers were monitored by Trypan Blue exclusion and cell counting using the Countess automated cell counter (Thermo Fisher Scientific). Cells were seeded at equal densities and grown to confluency over a period of 72 h without medium changes unless stated otherwise. Opti-MEM + GlutaMAX (Gibco) and lipofectamine RNAiMax (Invitrogen) were used for reverse transfection of endoribonuclease-prepared short interfering RNA (esiRNA) and short interfering RNA (siRNA) for 72 h. A list of the esiRNAs and siRNAs used in this study is provided in Supplementary Table 1. Where indicated, the following compounds were added to the medium: ddC (Sigma-Aldrich), BX795 (Sigma-Aldrich), VACV-70 70 bp oligonucleotides (InvivoGen) and tetracycline (Sigma-Aldrich). For deoxyribonucleoside supplementation during senescence, deoxyadenosine, deoxyguanosine, deoxycytosine and thymidine (Sigma-Aldrich) were added at 100 μM each to cultured cells and refreshed every 72 h.

Generation of KO and stable cell lines

Immortalized WT and Yme1l^−/− MEFs were described previously¹⁷. In brief, primary MEFs were isolated from Yme1l^loxP/loxP mice and immortalized using SV40 large T antigen-encoding plasmids. After transduction with Cre recombinase, individual clones were expanded and genotyped to identify WT (Yme1l^loxP/loxP) and Yme1l^−/− MEFs. WT, Mgme1^−/−, Sting1^mut/mut and Mgme1^−/−Sting1^mut/mut (DKO) primary MEFs were isolated from intercrossing of Mgme1^+/−Sting1^mut/mut mice and intercrossing of Mgme1^+/−Sting1^+/+ mice.

Establishment of cellular senescence

IMR90 fibroblasts were seeded on a diverse size of culture vessels with a density of 2,100 cells per cm² for DMSO (0.01%, v/v) and treated with decitabine (1 μM), or 6,500 cells per cm² for doxorubicin (300 nM) for TIS. The medium was replaced every other day. DMSO and decitabine were present in the medium at all times, whereas doxorubicin was washed out after the first medium change. The experiments were performed 10 days after the irradiation. Stress-induced senescence was achieved by exposing cells to X-ray irradiation at 20 Gy. Senescence was confirmed by the presence of p16 and p21, SA-β-Gal positivity (Cell Signaling, 9680, according to the manufacturer’s instructions) and the absence of genomic EdU incorporation (Thermo Fisher Scientific, C10337, according to the manufacturer’s instructions).

RT–qPCR

Cells were plated 1 × 10⁵ cells per ml in six-well plates and incubated for 72 h. Cells were scraped in cold PBS and pelleted. Total RNA from cells was isolated upon cell lysis using the NucleoSpin RNA isolation kit. cDNA was synthesized using the GoScript Reverse Transcription Mix (Promega) and qPCR was performed using PowerSYBR Green PCR Master Mix (Applied Biosystems). For each independent sample, qPCR was performed in technical duplicates. A list of the primer sequences used in this study is provided in Supplementary Table 2.

Animals and housing

This study was performed in accordance with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations (FELASA). The protocol was approved by the ‘Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen’ (reference numbers 81-02.04.2019.A378, 81-02.04.2020.A082, 81-02.04.2022.A453 and 2024-657). The mice were housed in standard individually ventilated cages (45 × 29 × 12 cm) under a 12 h–12 h light–dark schedule in controlled environmental conditions of 22 ± 2 °C and 50 + 10% relative humidity and fed a normal chow diet and water ad libitum. Generation of Mgme1^−/− mice was described previously⁴. Mgme1^−/−Sting1^mut/mut mice were generated by intercrossing those mice with Sting1^mut/mut (C57BL/6J-Sting1^gt/J, Jackson laboratory).

mtDNA extraction and Southern blot analysis

Total DNA and mtDNA was isolated from pulverized tissue/cellular pellets or purified mitochondria using Gentra Puregene Tissue Kit (Qiagen) or DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. DNA quantification was performed using the Qubit 1.0 fluorometer (Thermo Fisher Scientific). Then, 300–1,000 ng of DNA was digested with SacI (mouse) or BamH1 (human) restriction nucleases and DNA fragments were separated by neutral agarose gel electrophoresis, transferred to nitrocellulose membranes (Hybond-N+ membranes, GE Healthcare) and hybridized with α-P³²-dCTP-labelled probes). For estimation of rNMP content, DNA was treated with RNase H2 and separated by electrophoresis on 0.8% agarose gels under denaturing conditions. Southern blot signals were quantified using MultiGauge or ImageJ software and visualization and analysis was performed using InstantClue software⁴⁵. Primer and probe sequences used in this study can be found in Supplementary Table 3.

In organello replication

Freshly isolated mitochondria (300–500 μg) from MEFs were resuspended in 0.5 ml of incubation buffer (25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 10 mM K₂HPO₄, 0.05 mM EDTA, 5 mM MgCl₂, 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM Tris–HCl, pH 7.4, 1 mg ml⁻¹ fatty-acid-free BSA, 20 µCi α-³²P-dATP (3,000 Ci mmol⁻¹). Then, 50 µM each of dTTP, dCTP and dGTP was added in the incubation buffer where indicated. Incubation was carried out at 37 °C for 2 h on a rotating wheel. For the chase, reisolated mitochondria were incubated in 0.5 ml of incubation buffer supplemented with all four non-radiolabelled dNTPs (50 µM) for the indicated time. After incubation, mitochondria were pelleted at 9,000 rpm for 4 min and washed twice with wash buffer (10% glycerol, 10 mM Tris–HCl, pH 6.8, 0.15 mM MgCl₂). In the next step, DNA isolation and Southern blot analysis were performed as described above.

Detection of mtDNA in the cytosolic fraction using ddPCR or dPCR

For isolation of the cytosolic fraction, 2 × 10⁵ MEF cells were collected at 600g for 3 min and resuspended in 400 μl of isolation buffer (150 mM NaCl and 50 mM HEPES). After treating the cells with 25 μg ml⁻¹ digitonin in isolation buffer for 12 min on ice, the samples were centrifuged at 800g for 5 min at 4 °C. The supernatants were collected and further clarified by centrifugation at 25,300g for 10 min at 4 °C to obtain the cytosolic fraction¹⁰. The samples were normalized for protein concentration (BioRad). DNA was extracted from equal volumes of cytosolic fractions using the DNeasy DNA extraction kit (Qiagen) according to the manufacturer’s instructions. The mtDNA copy number was assessed using ddPCR (BioRad) using specific probes directed against different mtDNA regions. A list of the primer and probe sequences used in this study is provided in Supplementary Table 4.

The isolation of cytosolic and mitochondrial fractions from mouse tissues was performed as previously described⁴⁶. All of the samples were normalized to protein concentration using a DC protein assay (BioRad) and dPCR (Qiagen QiaCutie) was performed using the primer probe sets listed in Supplementary Table 4.

Nanostring analysis

Total RNA was isolated from tissues or cells using the NucleoSpin RNA isolation kit. The quality and quantity were validated using Nanodrop Analysis. Next, 200–500 ng of total RNA was applied for CodeSet hybridization. After 18 h of hybridization, the samples were loaded onto the Nanosting Cartridge and analysed in the nCounter SPRINT Profiler (nanoString) system according to the manufacturer’s instructions. ISGs and non-ISGs were filtered using the INTERFEROME database⁴⁷. The acquired data were analysed with nSolver software (nanoString). z-score intensities were calculated using the log₂-transformed intensities and clustering was performed using the Euclidean distance metric and the ‘average’ method. q values were calculated using unpaired two-tailed Student t-tests with multiple-comparison correction using the two-stage step-up method of Benjamini, Krieger and Yekutieli. q < 0.05 was considered to be significant.

SDS–PAGE and immunoblot analysis

Cells were washed with cold PBS and resuspended in ice-cold RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.05% sodium deoxycholate, 1 mM EDTA) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (PhosSTOP, Roche). The lysates were incubated with constant agitation for 30 min at 4 °C followed by centrifugation at 14,500g for 10 min. Total protein (20–50 μg) was separated using SDS–PAGE, followed by transfer to nitrocellulose membranes and immunoblotting with the antibodies listed in Supplementary Table 5.

Kidney histology

PAS and immunohistochemistry staining procedures for formalin-fixed paraffin-embedded tissue samples were essentially performed as previously described⁴⁸. CD3 (99940, Cell Signaling, 1:100) and B220 (CD45R, 550286, BD Pharmingen, 1:50) antibodies were used for immunohistochemistry DAB staining. In brief, 2 µm formalin-fixed paraffin-embedded sections of kidney tissue were prepared, stained, digitalized using the Ventana DP 200 slide scanner (Roche Diagnostics) and analysed using QuPath (v.0.4.4)⁴⁹. Finally, whole-kidney sections were analysed (staining artefacts such as tissue detachment were manually excluded). Cells were segmented using the built-in cell segmentation tool of QuPath and marker-positive cells were detected by thresholding. The assessment of glomerular sclerosis was conducted using a five-tier scoring system, with values of 0 representing normal glomeruli; 1 indicating mesangial dilatation or capillary wall thickening; 2 denoting segmental sclerosis in less than 25% of the glomerular tuft area; 3 indicating 25–50% of tuft area; and 4 indicating more than 50% of tuft area sclerosed. The mean score (0 to 4) and the percentage of sclerosed glomeruli (glomeruli with scores ≥ 2) were calculated.

Immunofluorescence and imaging

Cells were plated at a density of 4 × 10⁴ cells per coverslip. After incubation for 24 h, the medium was removed and cells were fixed on coverslips using 5% PFA/PBS (pH 7.4) for 15 min at 37 °C. After three quick PBS washes, autofluorescence was quenched using 50 mM NH₄Cl/PBS for 10 min at room temperature with slowly shaking. After two quick rinses with PBS, cells were permeabilized using 0.1% Triton X-100 in PBS for 15 min at room temperature and washed with PBS again. After blocking the coverslips for 20 min at room temperature using 10% FBS/PBS, the coverslips were incubated with primary antibodies (Supplementary Table 5) for 1 h or overnight and washed three times with PBS before incubation with secondary antibodies for 1 h in the dark. After incubation, cells were washed with PBS, dipped into double-distilled H₂O to remove salt residues and mounted using ProLong Gold (Invitrogen). Images were acquired using the Leica SP8-DLS laser-scanning confocal microscope equipped with an ×100 oil HC PL APO CS2 objective (numerical aperture (NA) 1.4), Leica DMI 6000 B wide-field fluorescence microscope equipped with a 100× oil HCX PL APO objective (NA 1.46) or Invitrogen EVOS FL Auto 2 microscope equipped with colour camera (to visualize positive X-Gal staining). Immunofluorescence images shown in Fig. 2d were acquired with a spinning-disk microscope. Stained cells were imaged using a 100× objective lenses (NA 1.4) on a Nikon Eclipse TiE inverted microscope with a four-channel integrated laser engine (ILE-400) using an Andor Dragonfly 500 spinning-disk system, equipped with a Zyla 4.2 PLUS sCMOS camera (Andor), coupled with Fusion software (Andor). Seven stacks of 0.2 μm each were acquired using the 100× objective. All images from a same experiment were acquired with the same parameters including exposure time and laser intensities. Images were compiled using ‘max projection’, and ‘smooth’ was applied once for representative images using the FIJI software.

HydEn-seq analysis

HydEn-seq and DNA libraries for sequencing were prepared as previously described⁴³. In brief, WT, Yme1l^−/− and Mgme1^−/− MEF cells were mapped by HydEn-seq by hydrolysing 1 μg DNA with 0.3 M KOH for 2 h at 55 °C. Then, 1 μg of DNA was digested or left untreated with 10 U of SacI (Ymel1^−/− and its WT control) or 10 U of HincII (Mgme1^−/− and its WT control), and the digests were purified with HighPrep PCR beads (Mag-Bio) before the KOH treatment. The libraries were then purified, quantified using the Qubit fluorometric instrument (Thermo Fisher Scientific) and 75-bp-end sequenced on an Illumina NextSeq 500 instrument to identify the location of the free 5′ ends. Breakpoints arising from restriction digestion were removed during data processing. Digested and undigested samples, which showed the same trend, were analysed together as independent replicates.

Library preparation and Illumina paired-end DNA sequencing

Library preparation and Illumina paired-end DNA sequencing were performed by the Cologne Center for Genomics (https://ccg.uni-koeln.de/technologies/next-generation-sequencing). mtDNA quality was verified using the Agilent TapeStation Genomic DNA ScreenTape and the Qubit BR kit. After fragmentation of 500 ng of gDNA to the desired insert size of >500 bp, the libraries were produced using the Illumina DNA PCR-Free Library Prep kit. After sequencing, the following run conditions were used (2 × 300 bp) on the Illumina NextSeq 2000 sequencing system, using the 1x NextSeq 2000, P1 FlowCell from Illumina. Each library was individually indexed with the barcodes provided and sequenced to 100 million reads. Reads were mapped to the mouse MT genome (GRCm39) with bwa (v.0.7.17; mem-T 19).

Protein digestion

Subcellular fraction proteomics

Mitochondria and total (cell pellet) were directly lysed in 4% SDS in 100 mM HEPES buffer (pH 8.5) at 70 °C for 10 min. Cytosolic fractions were subjected to acetone-based protein precipitation. The pellet was washed twice with 80% acetone and dried under the fume hood. The lysates were sonicated (Bandelin, Mini, 60 s, 1 s pulse, 1 s pause). Proteins were reduced (10 mM TCEP) and alkylated (20 mM CAA) in the dark for 45 min at 45 °C. The samples were subjected to an SP3-based digestion. Washed SP3 beads (SP3 beads (Sera-Mag), magnetic carboxylate modified particles (hydrophobic, GE44152105050250), Sera-Mag magnetic carboxylate modified particles (Hydrophilic, GE24152105050250) from Sigma-Aldrich) were mixed equally, and 3 µl of bead slurry was added to each sample. Acetonitrile was added to a final concentration of 50% and washed twice using 70% ethanol (V = 200 µl) on an in-house-manufactured magnet. After an additional acetonitrile wash (V = 200 µl), 5 µl digestion solution (10 mM HEPES pH 8.5) containing 0.5 µg trypsin (Sigma-Aldrich) and 0.5 µg LysC (Wako) was added to each sample and incubated overnight at 37 °C. Peptides were desalted on a magnet using 2 × 200 µl acetonitrile. Peptides were eluted in 10 µl 5% DMSO in LC–MS water (Sigma-Aldrich) in an ultrasonic bath for 10 min. Formic acid and acetonitrile were added to a final concentration of 2.5% and 2%, respectively. The samples were stored at −20 °C before analysis using LC–MS/MS.

LC and tandem MS-based proteomics

Subcellular fraction proteomics

Tryptic peptides (200 ng) were loaded onto an Evotip and analysed using the Evosep One LC (Evosep) system connected to a timsTOF Pro 2 (Bruker). The Evosep One method was 30 SPD (44 min gradient) using the EV1137 performance column (15 cm × 150 µm, 1.5 µm particle size) at 50 °C using a 20 µm Bruker emitter. The mass spectrometer was operated in DIA-PASEF mode. The DIA-PASEF method consisted of 24 m/z windows leading to a cycle time of 1.38 s. The other mass spectrometer parameters were set as follows: DIA m/z range, 480 to 1,000, the mobility (1/K₀) range was set to 0.895 to 1.21 V s cm⁻², and the accumulation and ramp time was 100 ms.

Data analysis

Raw files were analysed using Spectronaut (v.18.5.231110.55695)⁵⁰ in direct DIA mode using the UniProt (one sequence per gene) Mus musculus reference proteome (21,000 sequences, downloaded 2021). Trypsin/P was selected as the cleavage rule using a specific digest type. The minimal peptide length was set to 7 and a total of 2 missed cleavages were allowed. The peptide spectrum match, peptide and protein group false-discovery rate were controlled to 0.01. The mass tolerances were used with the default settings (Dynamic,1). The directDIA+ (deep) workflow was selected. The cross-run normalization was disabled. Peptide and protein group quantity was accessed by the sum of the precursor and peptide quantity, respectively. Mitochondrial proteins were identified using the MitoCarta 3.0 dataset⁵¹. The intensities were log₂-transformed and the median across the subcellular fractions was adjusted to the median of all samples of the respective fraction. The log₂-transformed intensities were scaled between 0 and 1 per protein group and visualized in a heat map using Instant Clue⁴⁵ by clustering the rows using the Euclidean distance metric and the ‘average’ method.

AEX–MS analysis of anionic metabolites

Extracted metabolites were resuspended in 100 µl of Optima LC–MS-grade water (Thermo Fisher Scientific), and transferred to polypropylene autosampler vials (Chromatography Accessories) before anion-exchange chromatography–MS (AEX–MS) analysis.

The samples were analysed using a Dionex ion chromatography system (ICS500+, Thermo Fisher Scientific) as described previously⁵². In brief, 5 µl of polar metabolite extract was injected in push partial mode, using an overfill factor of 2, onto the Dionex IonPac AS11-HC column (2 mm × 250 mm, 4 μm particle size, Thermo Fisher Scientific) equipped with a Dionex IonPac AG11-HC guard column (2 mm × 50 mm, 4 μm, Thermo Fisher Scientific). The column temperature was held at 30 °C, and the auto sampler was set to 6 °C. A potassium hydroxide gradient was generated using a potassium hydroxide cartridge (Eluent Generator, Thermo Fisher Scientific), which was supplied with deionized water. The metabolite separation was carried at a flow rate of 380 µl min⁻¹, applying the following gradient conditions: 0–3 min, 10 mM KOH; 3–12 min, 10 − 50 mM KOH; 12–19 min, 50–100 mM KOH; 19–21 min, 100 mM KOH; 21–22 min, 100–10 mM KOH. The column was re-equilibrated at 10 mM for 8 min.

For the MS analysis of metabolic pool sizes and the corresponding deoxy-/ribo- nucleotide ratios, the eluting compounds were detected in negative ion mode [M-H]⁻ using the multiple reaction monitoring mode on the Xevo TQS triple quadrupole mass spectrometer (Waters). The following settings were set: capillary voltage, 2.1 kV; desolvation temperature, 500 °C; desolvation gas flow, 800 l h⁻¹; collision cell gas flow, 0.15 ml min⁻¹. The detailed quantitative and qualitative transitions and retention times for the analysed nucleotides are summarized in Supplementary Table 6.

The obtained AEX–MS data analysis was performed by converting the raw data into mzML files using the MSConvert GUI from the ProteoWizzard suite⁵³. The obtained mzML files were analysed using the metabolomics software ElMaven⁵⁴. For relative quantification analysis, the area of the quantitative transition of each compound was extracted and integrated using a retention time tolerance of <0.1 min as compared to the independently measured reference compounds. Areas of the cellular pool sizes were normalized to the internal standards (¹³C₁₀ ATP, ¹⁵N₅ ADP or ¹³C₁₀⁵N₅ AMP), followed by a normalization to the protein content of the analysed cell sample.

Protein expression and purification

POLγA, POLγB and the TWINKLE DNA helicase were cloned and expressed as 6×His-tagged fusion proteins in Spodoptera frugiperda (Sf9) cells, according to previously published protocols⁵⁵. The SSBP1 gene encoding the human mitochondrial single-stranded DNA-binding protein (mtSSB) was cloned into the pET-17b vector in frame with a C-terminal 6×His-tag. The mtSSB protein was expressed in Escherichia coli and subsequently purified as previously reported⁵⁶.

In vitro DNA rolling circle replication

A 70-mer oligonucleotide (5′-T₄₂ATCTCAGCGATCTGTCTATTTCGTTCAT-3′) was annealed to single-stranded pBluescript SK(+) O_L⁵⁵, followed by one cycle of polymerization with KOD polymerase to produce a 4 kb double-stranded template with a preformed replication fork. The template (0.4 nM) was added to a reaction mixture (25 μl) containing 25 mM HEPES (pH 7.6), 10 mM DTT, 0.1 mg ml⁻¹ BSA, 4 mM ATP, all four dNTPs (2.5 µM or 10 µM, as indicated), 2 μCi α-[³²P]dCTP, 8 nM TWINKLE (calculated as a hexamer), 160 nM mtSSB (calculated as a tetramer) and 4 nM POLγA in complex with 6 nM POLγB (calculated as a dimer). GTP was added at increasing concentrations. In the 0 mM GTP control reaction, 10 mM MgCl₂ was included; for the other conditions, the MgCl₂ concentration was increased proportionally to match the GTP levels. The reactions were terminated after 60 min at 37 °C by the addition of 8 μl of alkaline loading buffer (18% (w/v) Ficoll, 300 mM NaOH, 60 mM EDTA (pH 8.0), 0.25% (w/v) bromophenol blue and 0.25% (w/v) xylene cyanol). The products were separated at 40 V for 20 h on a 0.8% denaturing agarose gel and visualized by autoradiography.

In vitro DNA replication on a single-stranded template

A 32-mer oligonucleotide [³²P]-labelled at the 5′ end (5′-CTATCTCAGCGATCTGTCTATTTCGTTCATCC-3′) was annealed to a single-stranded pBluescript SK(+) O_L plasmid. The reactions (25 µl) contained 0.4 nM of DNA template, 25 mM HEPES (pH 7.6), 10 mM DTT, 10 mM MgCl₂, 0.1 mg ml⁻¹ BSA, 160 nM mtSSB (calculated as a tetramer) and 4 nM POLγA in complex with 6 nM POLγB (concentration calculated as a dimer). The reactions contained 2.5 µM or 10 µM of dATP, dTTP, dGTP and dCTP. When indicated, ATP, CTP and UTP were added at increasing concentrations.

The reactions were incubated at 37 °C and stopped after 15 min with 5 µl stop buffer (90 mM EDTA, 6% SDS, 30% glycerol and 0.25% bromophenol blue). The products were separated on a 0.8% agarose gel with 0.5 μg ml⁻¹ ethidium bromide (EtBr) at 40 V in 1× TBE buffer for 18 h and visualized by autoradiography.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.