The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation – Nature

Cell culture

HeLa (CCL-2) cells were obtained from Sigma-Aldrich. HEK293T cells were a gift from D. Trono (EPFL), originally purchased from ATCC. U2OS cells, THP-1 cells and BJ-5ta cells were obtained from ATCC. CT26 mouse colorectal carcinoma cells were a gift from the D. Hanahan laboratory (EPFL). Primary human endothelial cells were obtained from a commercial supplier (Cell Biologics). HeLa and HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, 41965039) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco, 10270106), 100 IU ml⁻¹ penicillin–streptomycin (BioConcept, 4-01F00-H), 2 mM l-glutamine (Thermo Fisher Scientific, 25030024) and 1 mM sodium pyruvate (BioConcept, 5-60F00-H) at 37 °C under atmospheric O₂ and 5% CO₂. THP-1 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, 21875091) supplemented with 10% FBS, 1× penicillin–streptomycin–l-glutamine (Corning, 30-009-Cl) and 1× 2-mercaptoethanol (Gibco) at 37 °C under atmospheric O₂ and 5% CO₂, and differentiated with 10 μg ml⁻¹ phorbol-12-myristate-13-acetate (Sigma-Aldrich, P8139) for 2 days before the experiments. BJ-5ta cells were cultured in a mixed medium consisting of 80% DMEM (Thermo Fisher Scientific, 41965039) and 20% of medium 199 (Thermo Fisher Scientific, 41150087) supplemented with 10% FBS, 2 mM l-glutamine and 1× penicillin–streptomycin–l-glutamine. Primary human endothelial cells were cultured in complete human endothelial cell medium (Cell Biologics, H1168), according to the supplier’s instructions. CT26 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, 21875091) supplemented with 10% FBS, 100 IU ml⁻¹ penicillin–streptomycin (BioConcept, 4-01F00-H), 1 mM sodium pyruvate (BioConcept, 5-60F00-H) and 10 mM HEPES buffer (BioConcept, 5-31F00-H). CGAS-KO HeLa cells were reported previously³⁸. U2OS CGAS-KO cells were generated as described previously³⁸. CGAS-KO THP-1 cells were purchased from Invivogen. CT26 CGAS-KO cells were generated as described previously³⁸.

HeLa cells, displaying a functional cGAS–STING pathway, were used for most of the experiments. U2OS cells, expressing cGAS, were used to independently validate hits obtained from the siRNA screen. HEK293T cells were used for immunoprecipitation and ubiquitylation studies using transiently expressed tagged constructs. BJ-5ta cells, THP-1 cells and primary endothelial cells were used to ascertain the relevance of cGAS nuclear degradation by the CRL5–SPSB3 ubiquitin ligase across distinct cell types and primary human cells. Cell lines were repeatedly tested for mycoplasma by PCR. No method of cell line authentication was used.

Plasmids

For CRISPR–Cas9 plasmids, single guide RNAs (sgRNAs) targeting cGAS were designed using the web tool CRISPOR³⁹. sgRNAs targeting cGAS were cloned into the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene, 48138). pBABE-HA-ubiquitin was generated by inserting amplified HA-ubiquitin sequences from HA-ubiquitin (Addgene, 18712) into the pBABE vector. The pEFBos-cGAS-Flag, pEFBos-SPSB3-Flag and pEFBos-eGFP-Flag vectors were obtained by inserting cGAS, SPSB3 and eGFP sequences flanked by 5′ XhoI and 3′ NotI sites into the pEFBos vector. The pEFBos-based cGAS mutations (N513A/N514A, K427R/K428R and KRKKNN (K173E, R176E, K407E, K411A, N513A/N514A) and SPSB3 truncations (SPSB3ΔSOCS) were obtained by site-directed mutagenesis. The pTRIPZ-based cGAS and cGAS mutations (N513A/N514A, K427R/K428R and KRKKNN) were generated by inserting the coding sequences of cGAS flanked by 5′ XhoI and 3′ NotI sites into the pTRIPZ vector. The primers used for plasmid generation and sgRNA sequences are provided in Supplementary Table 2.

Generation of cell lines

To establish HeLa cells or U2OS cells with inducible expression of distinct cGAS constructs, corresponding CGAS-KO cells were infected with a pTRIPZ lentiviral vector carrying cGAS-GFP or cGAS-Dendra2 and a puromycin-resistance gene. Cells were selected with puromycin (1 μg ml⁻¹). To generate HeLa cGAS–GFP cells co-expressing PCNA–mCherry, HeLa cGAS–GFP cells were infected with a pRRL lentiviral vector (gift from D. Trono) carrying PCNA-mCherry and a blasticidin-resistance gene. Cells were selected using blasticidin (5 μg ml⁻¹). HeLa cells expressing doxycycline-inducible Flag–cGAS, Flag–cGAS(NN), Flag–cGAS(KK) or Flag–cGAS(KRKKNN) were generated from HeLa CGAS-KO cells by infection with a pTRIPZ lentiviral vector carrying the corresponding inserts and a puromycin-resistance gene. Cells were selected with puromycin (1 μg ml⁻¹).

Transfection

For plasmid transfection, cells plated in a six-well plate were transfected with plasmids and GeneJuice transfection reagent (Millipore, 70967) according to the manufacturer’s protocol. For siRNA transfection, 3 × 10⁴ cells were transfected with Lipofectamine RNAiMAX transfection reagent (Invitrogen, 13778075) and 40 pmol siRNA according to the manufacturer’s protocol and incubated for 3–5 days. The medium containing transfection reagents was replaced with fresh medium 6 h after transfection. siRNA targeting SPSB3 (s40519, s40520) and CUL5 (s15588) and a negative control siRNA (4390847) were purchased from Thermo Fisher Scientific. Sequences of siRNAs are provided in Supplementary Table 2. For mRNA transfection, 2 × 10⁵ cells per well CT26 Cgas-KO mouse colorectal carcinoma cells or HeLa CGAS-KO cells were seeded into 12-well plates. Then, 500 ng of mRNA prediluted in Opti-MEM (Thermo Fisher Scientific) was mixed with 1.8 μl Lipofectamine RNAiMAX (Thermo Fisher Scientific; prediluted 1:20 in OptiMEM) for each well. After 15 min incubation at room temperature, mRNA mixture was added to cells. After 6 h, the medium was changed to remove mRNA. Cells were collected on day 0, 1, 2 or 4. mRNAs were generated by in vitro transcription at the mRNA platform of the University of Zürich (CH) as described previously⁴⁰. Sequences of mRNAs are provided in Supplementary Table 2.

RNAi screen targeting factors of the UPS

To identify regulators of nuclear cGAS stability, HeLa cells expressing doxycycline-inducible cGAS–GFP were treated with siRNAs targeting 972 UPS genes (three independent siRNAs per target) using the A30140 Silencer Select Human Ubiquitin siRNA library (Thermo Fisher Scientific) (Supplementary Table 1). Each plate (96-well PhenoPlate (PerkinElmer)) comprised 8 control wells, including 2 wells with negative control siRNA, 2 wells with a cGAS-targeting siRNA, and 4 wells containing epoxomicin (100 ng ml⁻¹). For reverse transfection, 5 μl of siRNA (200 nM in Opti-MEM (Thermo Fisher Scientific)) was mixed with 5 μl Lipofectamine RNAiMAX (Thermo Fisher Scientific) (prediluted 1:20 in OptiMEM) inside each well. After 15 min incubation at room temperature, 3,500 cells were added to each well in 100 μl complete DMEM (10% FBS, 1% penicillin–streptomycin–glutamine solution, 1 μg ml⁻¹ doxycycline). After 48 h, epoxomicin was added to the respective control wells. After incubation overnight (day 3 after reverse transfection), cells were washed with PBS and fixed by addition of a paraformaldehyde 4% solution in PBS supplemented with Hoechst (1 μg ml⁻¹). Fixed cells were stored in PBS at 4 °C before imaging. Imaging of cells (entire well; 21 images at ×10 magnification) was performed using the IN Cell Analyzer 2200 microscope (GE Healthcare Life Sciences). Analysis of nuclear cGAS–GFP levels was performed using the CellProfiler software. In brief, each nucleus was identified by its Hoechst signal and intranuclear cGAS–GFP MFI and the integrated fluorescence intensity was quantified. The final results were obtained by calculating the relative values for MFI and integrated fluorescence intensity relative to the controls (cGAS-targeting siRNA = −1; negative control siRNA = 0; epoxomicin = 1) and data were visualized by blotting relative MFI levels against integrated fluorescence intensity levels. Genes were defined as hits if their relative fluorescence intensity values deviated from the negative control siRNA by more than 3 s.d.

Live-cell imaging

HeLa cells expressing cGAS–GFP were plated in Falcon 96-well microplates (black; clear flat-bottom; tissue-culture-treated) (Corning) at a density of 3,500 to 7,500 cells per well in complete FluoroBrite (Thermo Fisher Scientific) DMEM (10% FBS, 2 mM l-glutamine, 1% penicillin–streptomycin and 1 μg ml⁻¹ doxycycline). The next day, cells were transfected or not with siRNAs and incubated for 2 days. Drug treatment, including epoxomicin (200 nM), bortezomib (100 nM) and leptomycin B (80 nM), was performed 1 h before live-cell recording. Live-cell imaging of cGAS(WT)–GFP was performed using a confocal Leica SP8 Inverted microscope equipped with an HC PL APO ×63/1.40 NA oil-immersion objective and HyD detectors, operated with the Leica LAS X software. For experiments with cGAS(WT)–GFP and cGAS(NN) or cGAS–Dendra2, live-cell imaging was performed on the Zeiss LSM980 Inverted microscope (Carl Zeiss) equipped with a motorized, heated stage and a full incubation chamber maintaining 37 °C and 5% CO₂ and operated with the Zeiss ZEN software. Images were acquired every 3–5 min. Image analysis was performed using Fiji (v.2.3.0) on a nucleus-by-nucleus basis and data were further quantified using GraphPad PRISM 9 (v.9.3.1). In experiments using cGAS–Dendra2, Dendra2 was photoactivated before live-cell imaging using the 405 nm laser (700 Hz; laser power: 7.5%) to scan the same region of interest four times (frame average). For cell cycle phase analysis, a single cell nucleus was tracked over time using PCNA–mCherry as a mask for the nucleus, while recording the cGAS–GFP nuclear intensity. A change in PCNA–mCherry signal from a diffuse pattern to a dot-like pattern was used to define the transition from G1 to S phase of the cell cycle, whereas the reverse change was used to define the transition from S to G2 phase of the cell cycle.

Immunofluorescence and confocal imaging

Cells were plated in CellCarrier-96 Ultra Microplates (Perkin Elmer, 6055302) at a density of 10,000 cells per well with at least 5 h incubation for adherence of the cells. For immunofluorescence, cells were washed once with PBS, then cells were fixed by adding paraformaldehyde 4% in CBS buffer (10 mM MES pH 6.9, 138 mM KCl, 2 mM MgCl₂, 2 mM EGTA) for 5–10 min at room temperature. Cells were washed three times for at least 5 min in PBS before blocking for 1–2 h at room temperature with PBS supplemented with 0.1% (v/v) Triton X-100 and 5% (v/v) heat-inactivated FBS and incubating overnight at 4 °C with the primary antibodies diluted in staining solution (PBS supplemented with 0.1% (v/v) Triton X-100) and 1% (w/v) bovine serum albumin (Sigma-Aldrich, A7906)). A list of the antibodies and the concentrations used is provided in Supplementary Table 2. The cells were washed with PBS three times for 5 min and incubated for 1 h at room temperature in secondary antibodies diluted in staining solution. From then on, the plate was protected from light. Cells were washed twice (5 min each) in PBS and incubated for 30–60 min in Hoechst 33342 (Sigma-Aldrich, B2261) 0.2 μg ml⁻¹ in PBS. Cells were kept in 100 μl PBS per well and either imaged directly or kept at 4 °C until imaging. Imaging was performed using the Zeiss LSM 980 Inverted microscope (Carl Zeiss) using the Plan-Apochromat ×63/1.40 NA oil-immersion objective. Image analysis and quantifications were performed using Fiji (v.2.3.0).

Quantification of cGAMP levels by ELISA

cGAMP levels were quantified as previously described¹⁵. In brief, cells were collected by trypsination (trypsin-EDTA (0.05%), Life Technologies). Cell pellets were lysed in RIPA lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% (w/v) sodium deoxycholate, 0.03% (v/v) SDS, 0.005% (v/v) Triton X-100, 5 mM EDTA, 2 mM sodium orthovanadate and cOmplete Protease Inhibitor Cocktail (Roche). Lysed cells were centrifuged for 10 min at 18,200 g and 4 °C. The supernatant was used for the cGAMP ELISA assay (Cayman 2′3′-cGAMP ELISA kit, 501700) according to the manufacturer’s instructions. The protein concentration in the supernatant was measured using the BCA Pierce Protein assay kit and was used to normalize cGAMP levels.

QIBC and analysis

Cells were plated in CellCarrier-96 Ultra Microplates (Perkin Elmer, 6055302) at a density of 10,000 cells per well with 24 h incubation for adherence of the cells. For experiments with cGAS–GFP cells, 1 μg ml⁻¹ doxycycline was added for 24 h before fixation. For experiments with drug treatment, epoxomicin (200 nM) or MLN4924 (1 μM) was added for 16 h or 8 h before fixation. For experiments with siRNA knockdown, cells were transfected with Lipofectamine RNAiMAX transfection reagent (Invitrogen, 13778075) according to the manufacturer’s protocol followed by 5 days of incubation before fixation. To define the population of cells in a different cell cycle phase, EdU and DNA were fluorescently labelled using the Click-IT EdU Imaging Kit with Alexa Fluor 594 Azide (Invitrogen, C10086) and DAPI according to the manufacturer’s instructions. Cells were then stained with primary and secondary antibodies as described before.

High-content images were acquired on the fully automated Operetta CLS (PerkinElmer) system at ×20 operated with Harmony (PerkinElmer) software. Analysis of fluorescence intensities was performed using CellProfiler software. Nuclear intensity measurements were determined within a nuclear mask generated from DAPI staining. Cytoplasmic intensity measurements were determined within a ringed mask outside the nucleus and inside the cell border generated from the digital phase contrast channel. The background was determined by LowerQuartileIntensity and subtracted from the mean intensity of the given fluorescent marker. Colour-coded scatter plots were generated using KNIME v.4.7.4 (KNIME) with custom scripts. The DNA content was determined using the integrated intensity of DAPI signal. The S phase cell population was determined by the mean intensity of EdU signal. The G2/M phase cell population was determined by the DNA content and the M phase population was then extracted by the mean intensity of DAPI signal, which was further confirmed by anti-H3pS10 staining. Finally, the intensities of different populations were collected and analysed using GraphPad PRISM 9 (v.9.3.1).

Antibodies

The following primary antibodies were used: mouse monoclonal anti-vinculin (hVIN-1; Sigma-Aldrich, V9264, immunoblot, 1:5,000), rabbit monoclonal anti-GAPDH (14C10; Cell Signaling Technology, 2118, immunoblot, 1:3,000), mouse monoclonal anti-Flag (M2; Sigma-Aldrich, F1804, immunoblot, 1:5,000), rabbit monoclonal anti-ISG15 (EPR24482-49; Abcam, ab285367, immunoblot, 1:1,000), rabbit polyclonal anti-ISG15 (Cell Signaling Technology, 2743S, immunoblot, 1:1,000), rabbit polyclonal anti-cGAS (Novus, NBP3-16666, immunoprecipitation, 1:50), rabbit monoclonal anti-cGAS (D3O8O; Cell Signaling Technology, 31659, immunoblot, 1:1,000), rabbit monoclonal anti-cGAS (D1D3G; Cell Signaling Technology, 15102, immunoblot, 1:1,000), rabbit monoclonal anti-cGAS (E5V3W; Cell Signaling Technology, 79978, immunoblot, 1:1,000; immunofluorescence, 1:200), mouse monoclonal anti-ubiquitin (P4D1; Santa Cruz, sc-8017, 1:500), rabbit monoclonal anti-ubiquitin (linage-specific K48; EP8589; Abcam ab140601, immunoblot, 1:200), rabbit polyclonal anti-SPSB3 (Aviva, ARP71676_P050, immunofluorescence 1:100, immunoblot, 1:1,000), rabbit polyclonal anti-H3pS10 (Sigma-Aldrich, 06-570, immunofluorescence 1:200), rabbit polyclonal anti-cullin-5 (Abcam, ab264284, immunofluorescence 1:100), mouse monoclonal anti-His-tag (27E8; Cell Signaling Technology, 2366, immunoblot, 1:1,000), rabbit monoclonal anti-H2B (Abcam, 52484, immunoblot, 1:5,000). The following HRP-conjugated secondary antibodies were used: donkey anti-rabbit IgG (H+L)-HRP (Jackson ImmunoResearch, 711-036-152, immunoblot: 1:5,000) and donkey anti-mouse IgG (H+L)-HRP (Jackson ImmunoResearch, 715-036-151, immunoblot: 1:5,000). The following fluorescence-conjugated secondary antibodies were used: goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568-conjugated (Invitrogen, A-11011, immunofluorescence 1:800), goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488-conjugated (Invitrogen, A-11008, immunofluorescence 1:800), Click-iT EdU Imaging Kit with Alexa Fluor 647 (Thermo Fisher Scientific, C10086). A list of antibodies is provided in Supplementary Table 2.

RT–qPCR analysis

Cells were lysed in the RLT buffer (Qiagen). RNA was extracted according to the manufacturer’s protocol (Qiagen RNeasy Mini Kit). RNA was reverse transcribed using PrimeScript RT Reagent Kit (Takara) and analysed by RT–qPCR in triplicates using the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific). The qPCR reactions were run on the QuantStudio 7 Real-Time PCR system (Thermo Fisher Scientific). GAPDH was used as the housekeeping gene for normalization. Primer sequences are provided in Supplementary Table 2.

Cell fractionation

Cells from a 10 cm² dish were lysed with cold 400 μl buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 20% Triton X-100) on ice for 5 min. Cytosolic fractions were collected by centrifugation at 3,500 rpm, 4 °C for 5 min. Pellets were washed twice with buffer A before being resuspended with 400 μl buffer B (3 mM EDTA, 0.2 mM EGTA) on a rotator at 4 °C for 30 min. The soluble nuclear fractions were collected by centrifugation at 4,000 rpm, 4 °C for 5 min. The chromatin pellets were then washed with buffer B twice until they became almost invisible before being lysed with salt buffer (50 mM Tris pH 7.5, 300 mM NaCl, 1% Triton X-100, 1 mM DTT) and boiled with 1× loading buffer for 20 min.

Immunoblotting and immunoprecipitation

Cells were collected, quickly rinsed with 1× PBS and lysed in lysis buffer (20 mM Tris pH 7.4, 0.5% Triton X-100, 150 mM NaCl, 1.5 mM MgCl₂, 2 mM EGTA, 2 mM DTT and 1× cOmplete Protease Inhibitor Cocktail (Roche)) on ice for 30 min and centrifuged at 12,000 rpm, 4 °C for 10 min. The supernatants were boiled with 4× loading buffer (200 mM Tris pH 6.8, 8% SDS, 40% glycerol, 0.4 M DTT, 0.4% bromophenol blue) for 10 min. Proteins were resolved by SDS–PAGE using SurePAGE precast gels (GenScript) and transferred to nitrocellulose membranes using the Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit (Bio-Rad) according to the manufacturer’s instructions. Membranes were blocked with 3% skimmed milk in PBST (PBS with 0.05% Tween-20) at room temperature for 1 h and then incubated with the primary antibody (diluted in PBST) at 4 °C overnight. After washing in PBST, membranes were incubated with the secondary antibody at room temperature for 1 h. Membranes were washed with PBST, visualized with western blotting detection reagent (Bio-Rad) and imaged using the ChemiDoc XRS Bio-Rad Imager and Image Lab Software.

For immunoprecipitation, cells were seeded into six-well plates and were transfected with the indicated plasmids. Then, 16 h after transfection, cells were lysed in IP lysis buffer (PBS supplemented with 1% TEGITOL solution, 0.5% sodium deoxycholate, 1× cOmplete Protease Inhibitor Cocktail (Roche) and 1 μl benzonase) on ice for 30 min and centrifuged at 12,000 rpm 4 °C for 10 min. The supernatants were transferred into new tubes and mixed with anti-Flag M2 magnetic beads (Sigma-Aldrich, M8823) or ChromoTek GFP-Trap agarose (Proteintech) at 4 °C overnight on a rotator. After 3–6 washes with IP wash buffer (PBS supplemented with 300 mM NaCl), beads or agaroses were boiled with 1× loading buffer for 10 min. Gels were loaded with 20 μl samples, and then analysed using SDS–PAGE and immunoblotting. Full scans of the immunoblots are shown in Supplementary Fig. 1.

Cycloheximide chase experiments

After treatment of cGAS–GFP-expressing HeLa cells with 500 nM MLN4924 for 1 h or siSPSB3/siControl for 48 h, cells were treated with cycloheximide (40 μg ml⁻¹) for 0, 2, 4 and 6 h before collection. Cells were fractionated using the commercial subcellular protein fractionation kit (Thermo Fisher Scientific, 78840). The fractionated samples were diluted in Laemmli buffer and boiled for 20 min at 98 °C. Expression of the indicated proteins was determined using immunoblotting.

Protein expression and purification

Halo-tagged human cGAS WT, Halo-tagged cGAS catalytic domain (amino acids 155–522) and the corresponding substitutions in the pET-28 vector were expressed in BL21 (DE3) bacteria (Sigma-Aldrich, CMC0014). A single colony was inoculated in a culture flask with 100 ml LB with kanamycin (50 μg ml⁻¹) and incubated with shaking (200 rpm, Infors-HT Multitron) at 37 °C overnight as preculture. Large-scale expression of the protein was started the next day by pouring 100 ml of the preculture in a 5 l Erlenmeyer flask containing 2 l LB with kanamycin (50 μg ml⁻¹). The cells were grown until the optical density at 600 nm reached 0.7. Expression was then induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM while transferring the culture to an 18 °C shaking incubator overnight. Bacteria were collected by centrifugation, solubilized in HisTrap buffer A (20 mM HEPES, 500 mM NaCl, 20 M imidazole, 1 mM DTT and 5% glycerol, pH 7.5) supplemented with 4-(2-aminoethyl)-benzolsulfonylfluoride-hydrochloride (AEBSF) and cOmplete protease inhibitor (Roche), lysed by sonication, cleared by centrifugation at 20,000g and then passed through a 5 ml nickel-immobilized metal-affinity chromatography column (Cytiva, HisTrap HP, 17524802) on an fast protein liquid chromatography (FPLC) system. The protein of interest was eluted with buffer B (20 mM HEPES, 500 mM NaCl, 500 mM imidazole, 1 mM DTT and 5% glycerol, pH 7.5). Halo-tagged cGAS was then purified by SEC through the Superdex 75 Hiload 16/600 column (Cytiva 28-9893-33). For untagged cGAS, TEV enzyme was mixed with Halo-tagged cGAS overnight, and untagged cGAS was then purified by SEC through the Superdex 75 Hiload 16/600 column (Cytiva 28-9893-33). Halo-tagged mouse cGAS was prepared using the same procedure. All mutants were generated using a PCR-based technique with appropriate primers and confirmed by DNA sequencing.

The cDNA of human SPSB3 (amino acids 83–326) or its corresponding mutant was cloned into a pET-28 vector with an N-terminal His6-SUMO tag. SPSB3 was expressed in BL21 (DE3) (Sigma-Aldrich, CMC0014). A single colony was inoculated into a culture flask with 400 ml LB with kanamycin (50 μg ml⁻¹) and incubated with shaking (200 rpm, Infors-HT Multitron) at 37 °C overnight as preculture. Large-scale expression of the complex (8 l in total: 2 l in four 5 l Erlenmeyer flasks) was started the next day by pouring 100 ml of preculture into 2 l of Auto Induction Media Terrific Broth (Formedium, AIMTB0210) with kanamycin (50 μg ml⁻¹). The flasks were incubated with shaking at 37 °C for 6 h, then incubated at 18 °C overnight. The cells were then collected by centrifugation (4,000g, 15 min). The cell pellet of a 2 l expression culture was transferred into a Falcon 50 ml tube. The 2 l expression cells were solubilized in PBS with 1 mM DTT, 1 mM EDTA and 2% glycerol at pH 7.5, supplemented with AEBSF and cOmplete protease inhibitors (Roche), then lysed by sonication. The cell lysate was clarified by centrifugation followed by 0.45 μm filtration. The supernatant was first purified on a 5 ml nickel-immobilized metal-affinity chromatography column (Cytiva, HisTrap HP, 17524802) on a FPLC system (Cytiva Aktä Pure). ULP1 cleavage was performed at 4 °C overnight in a 3,500-Da molecular weight cut-off dialysis tube against PBS with 5% glycerol at pH 7.5. To remove the ULP1 protease as well as free SUMO, the sample was passed through a HisTrap and washed with PBS with 20 mM Imidazole. The flowthrough was collected and concentrated using the Superose 6 HiLoad 16/600 SEC (Cytiva, 29323952) equilibrated in PBS. All mutants were generated using a PCR-based technique with appropriate primers and confirmed by DNA sequencing.

Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025). In brief, the human UBE2R1 (UniProt: P49427) coding sequence was cloned into the pET-28-His-thrombin vector to enable expression as an N-terminal His-thrombin fusion protein; the human UBE2L3 (UniProt: P68036) coding sequence was cloned into the pGEX-4T-1 vector to enable expression as an N-terminal GST-fusion protein; the human ARIH2 (UniProt: O95376) coding sequence was cloned into the pGEX-4T-1 vector to enable expression as an N-terminal GST-fusion protein. After transformation of BL21(DE3) competent cells, protein expression (UBE2R1, UBE2L3, ARIH2) was induced by addition 1 mM IPTG when the optical density at 600 nm reached 0.6, and bacteria were cultured at 18 °C overnight. For His-tagged proteins, cell pellets were collected and suspended in lysis buffer (20 mM imidazole, 50 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 5 mM BME) supplemented with cOmplete protease inhibitors (Roche). After sonication, the cell lysates were clarified by centrifugation at 20,000g for 30 min. The supernatants were collected and incubated with Ni-NTA agarose at 4 °C for 2 h. Resins were washed and bound protein was eluted by 250 mM imidazole in buffer (50 mM Tris pH 8.0, 300 or 400 mM NaCl, 5% glycerol, 5 mM BME). To remove tags, TEV protease or thrombin was added to the eluates and untagged proteins were further purified by gel filtration with Superdex 75 increase 10/300 column (Cytiva, 29148721) equilibrated with buffer (25 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 1 mM DTT). Protein purify was checked by SDS–PAGE electrophoresis and pure fractions were pooled, flash-frozen in liquid nitrogen and stored at −80 °C until further use.

For GST-tagged proteins, cell pellets were collected and suspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 5 mM BME) supplemented with cOmplete protease inhibitors (Roche). After sonication, the cell lysates were clarified by centrifugation at 20,000g for 30 min. The supernatants were collected and incubated with Glutathione Sepharose Resins followed by washing, and the bound protein was eluted using 15 mM GSH in lysis buffer. Removal of GST tag was facilitated by adding thrombin to the eluates and the untagged protein was further purified by a second Glutathione Sepharose column followed by gel filtration with the Superdex 75 increase 10/300 column equilibrated with 25 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol and 1 mM DTT. The fractions containing pure protein were pooled, flash-frozen in liquid nitrogen and stored at −80 °C until further use.

BLI analysis

BLI analyses were performed at 25 °C using the GatorPrime biosensor system (GatorBio) with anti-His probes (GatorBio, 160009). cGAS or cGAS mutants (50 μg ml⁻¹) were immobilized onto the anti-His biosensor for 1 min. The tips were washed with PBS buffer for 2 min to obtain a baseline reading, then the biosensors were dipped into wells containing the various concentrations of SPSB3 or its mutant for 5 min. Then, a 10 min buffer wash was performed to allow the dissociation of molecules from the sensor. Data analysis was performed with GraphPad PRISM 9 using a standard 1:1 binding model. Three independent experiments were performed for each sample.

Cryo-EM data acquisition

A total of 1 mg cGAS(WT)-Halo-SPSB3-ELOBC or 1 mg cGAS(site C)-Halo-SPSB3-ELOBC complex was incubated with 1 mg recombinant mononucleosome core particle (Active motif, 81770) for 30 min on ice in PBS. Fractionations corresponding to the complex were collected on the Supedex 200 increase 10/300 GL column (Cytiva) in PBS and concentrated to 1 mg ml⁻¹. Aliquots of 3 μl of the cGAS–Halo–SPSB3–ELOBC–nucleosome complexes were loaded onto glow-discharged holey carbon grids (Electron Microscopy Sciences, CF-2/2-3Cu-50, C-Flat, Cu, R 2/2, 300 mesh). Grids were blotted for 4 s and plunge-frozen in liquid ethane using the Vitrobot at 4 °C with 100% humidity. The grids were screened for particle presence and ice quality on a the TFS Glacios microscope (200 kV), and the grids with the best quality were transferred to a TFS Titan Krios G4. Cryo-EM data were collected using the TFS Titan Krios G4 transmission electron microscope (TEM), equipped with a Cold-FEG on the Falcon IV detector in electron-counting mode. Falcon IV gain references were collected just before data collection. Data were collected with TFS EPU v.2.12.1 using the aberration-free image shift protocol (AFIS), recording eight micrographs per ice hole. Videos were recorded at ×96,000 magnification, corresponding to a pixel size of 0.83 Å at the specimen level, with defocus values ranging from −0.8 to −1.8 μm. Exposures were adjusted automatically to 60 e⁻ Å⁻² total dose, resulting in an exposure time of approximately 5 s per video. In total, 9,855 micrographs in EER format were collected for the cGAS WT complex and 15,141 micrographs in EER format were collected for the cGAS(site C) sample.

Cryo-EM data processing

Motion correction was performed on raw stacks without binning using the cryoSPARC implementation of motion correction. For the cGAS(WT)–Halo–SPSB3–ELOBC–nucleosome complex, a total of 972,457 particles was template-based automatically picked and particles were binned by a factor of 4. Two rounds of two-dimensional (2D) classification were performed, resulting in a particle set of 378,280 particles. Selected particles resulting from the 2D classification were used for ab initio reconstruction with two classes, and the one containing the nucleosome shape was selected as the initial model. A hetero-refinement was performed on three repeating initial models on the resulting 2D particles. A class with clear cGAS density was selected, including 69,435 particles. To deal with the sample heterogeneity, a mask on a single nucleosome and cGAS–SPSB3–ELOBC was generated for masked 3D-variability analysis with three modes. A mode showing variability on SPSB3 was selected for clustering with ten classes, among which, four classes containing SPSB3–ELOBC density were selected and re-centred and re-extracted. The particles were processed for iterative CTF refinement and non-uniform refinement in cryoSPARC to 4.30 Å. The reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion. For the cGAS(site C)–Halo–SPSB3–ELOBC–nucleosome complex, a total of 5,257,474 particles was template-based automatically picked and particles were binned by a factor of 4. Two rounds of 2D classification were performed, resulting in a particle set of 1,400,892 particles. Selected particles resulting from the 2D classification were used for ab initio reconstruction with two classes, and the one containing the nucleosome shape was selected as the initial model, including 1,183,961 particles. A non-uniform refinement refines the map to 7.03 Å. To deal with the sample heterogeneity, a mask on the single nucleosome and cGAS–SPSB3–ELOBC was generated for masked 3D-variability analysis with three modes. A mode showing variability on SPSB3 was selected for clustering with 20 classes, among which, 9 classes containing SPSB3 density were selected. Then, a mask on cGAS–SPSB3–ELOBC was generated and used for local refinement, followed by a masked 3D-variability analysis with three modes with the same mask. A mode showing variability on ELOBC was selected for clustering with 20 classes, among which, seven classes containing ELOBC density were selected, re-centred and re-extracted. The particles were processed for iterative CTF refinement and non-uniform refinement in cryoSPARC to a resolution of 2.75 Å. A local refinement on cGAS–SPSB3–ELOBC refined the map to 3.51 Å. The reported resolutions are based on the gold-standard FSC 0.143 criterion. Local-resolution variations were estimated using cryoSPARC⁴¹.

Model building and refinement

The nucleosome–cGAS–SPSB3–ELOBC model was generated using a published nucleosome cGAS structure (PDB: 6X59 (ref. ¹¹), 6Y5E (ref. ¹⁰) and 4LEV (ref. ⁴²)). Direct prediction of the cGAS–SPSB3–ELOBC complex failed to find SPSB3 interacting with cGAS by Alphafold2. The SPSB3–ELOBC model from AlphaFold2 was docked into the cryo-EM map in ChimeraX³⁷ and tuned by ISOLDE⁴³ and Coot⁴⁴. The whole model was then refined in PHENIX⁴⁵. Several loop regions of cGAS and SPSB3 were manually adjusted to fit into the map using Coot. The model was refined in real space again in PHENIX. All structure figures were made using UCSF Chimera⁴⁶ and UCSF ChimeraX³⁷.

Sequence alignments

A total of 150 human cGAS/SPSB3 orthologues in vertebrates were individually aligned and downloaded in ClustalW format from the Ensembl database. The sequence logo was derived from WebLogo (https://weblogo.berkeley.edu) on the indicated alignment regions. The human SPSB1/2/3/4 sequence as well as their percentage identity matrix analysis were downloaded from UniProt. Geneious Prime software was used to generate the sequence alignments shown in Extended Data Figs. 5d and  8c.

In vitro ubiquitylation assays

Human cGAS ubiquitylation assay was performed at 37 °C for 40 min. Reactions (20 μl) consisted of 50 μM ubiquitin, 100 nM UBE1, 1 μM UBE2R1, 0.35 μM CUL5–RBX2–NEDD8 complex, 0.7 μM SPSB3–ELOBC complex and 1 μM cGAS or cGAS mutants in buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 2.5 mM MgCl₂, 1 mM ATP, 2 mM DTT). If needed, 1 μM UBE2L3 and 300 nM ARIH2 were also added to the reaction. The reactions were initiated by ATP and quenched with 5× SDS–PAGE loading buffer after 40 min. Ubiquitylated cGAS products were separated on 4–20% SurePAGE (GenScript) and were probed with cGAS monoclonal antibodies (Cell Signaling, E5V3W). The ubiquitylation assay for mouse cGAS was slightly modified: 200 nM UBE1, 2 μM UBE2L3 and 600 nM ARIH2 were added to the reaction and the incubation time was extended to 3 h at 37 °C. Ubiquitylated mouse cGAS products were probed with His-tag monoclonal antibodies (Cell Signaling, 27E8).

Identification of ubiquitylation sites on cGAS

To map lysine residues on cGAS modified by ubiquitin, in vitro ubiquitylation reactions were performed in the presence of UBE2L3 and ARIH2. After SDS–PAGE electrophoresis, the gel was stained with Coomassie Blue and putative ubiquitylated cGAS bands were excised for MS analysis. MS-based proteomic experiments were performed at the Proteomics Core Facility of the School of Life Sciences at EPFL. The selected gel pieces were excised and washed twice with 50% ethanol in 50 mM ammonium bicarbonate (Sigma-Aldrich) for 20 min and dried by vacuum centrifugation. Proteins were reduced with 10 mM dithioerythritol (Merck-Millipore) for 1 h at 56 °C followed by a washing–drying step as described above. Reduced proteins were alkylated with 55 mM iodoacetamide (Sigma-Aldrich) for 45 min at 37 °C in the dark followed by a washing–drying step as described above. Proteins were digested overnight at 37 °C using MS-grade Trypsin Gold (Promega) at a concentration of 12.5 ng μl⁻¹ in 50 mM ammonium bicarbonate supplemented with 10 mM CaCl₂. The resulting peptides were extracted in 70% ethanol, 5% formic acid (Merck-Millipore) twice for 20 min, dried by vacuum centrifugation and stored at −20 °C until further analysis.

LC–MS/MS analysis

Peptides were desalted on C18 StageTips and dried by vacuum centrifugation before liquid chromatography coupled with tandem MS (LC–MS/MS) injections⁴⁷. The samples were resuspended in 2% acetonitrile (Biosolve), 0.1% formic acid and nano-flow separations were performed on the Dionex Ultimate 3000 RSLC nano UPLC system (Thermo Fischer Scientific) online connected with an Q Exactive Orbitrap Mass Spectrometer (Thermo Fischer Scientific). A capillary precolumn (Acclaim Pepmap C18, 3 μm, 100 Å, 2 cm × 75 μm inner diameter) was used for sample trapping and cleaning. A 50 cm long capillary column (75 μm inner diameter; in-house packed using ReproSil-Pur C18-AQ 1.9 μm silica beads) was then used for analytical separations at 250 nl min⁻¹ over 90 min biphasic gradients. The mobile phases were as follows: A, 2% acetonitrile, 0.1% formic acid in water; and B, 90:10 (v/v) acetonitrile:water, 0.1% formic acid. The mass spectrometer was operated in positive data-dependent acquisition mode, and the full MS range was from 300 to 2,000 m/z. The 12 most intense ions were isolated in the quadrupole and fragmented under high-energy collisional dissociation with a normalized collision energy of 27% with a 30 s exclusion list. Precursor and fragment ions were measured at a resolution of 70,000 and 17,500 (at 200 m/z), respectively. Only ions with charge states of 2 and higher were fragmented with an isolation window of 1.2 m/z.

Raw data were processed using SEQUEST, MS Amanda⁴⁸ and MS Fragger⁴⁹ in Proteome Discoverer 2.5 against the provided sequences on a Homo sapiens proteome background. Enzyme specificity was set to trypsin and a minimum of six amino acids was required for peptide identification. Up to two missed cleavages were allowed and a 1% FDR cut-off was applied both at the peptide and protein identification levels. For the database search, carbamidomethylation was set as a fixed modification, whereas oxidation (M), acetylation (protein N-term), PyroGlu (N-term Q) and phosphorylation (S, T, Y) and K-GG were considered as variable modifications. Data were further processed and inspected in Scaffold v.5.1.0 (Proteome Software).

Virus infection experiments

GFP-expressing virus strains were a gift from F. Schmidt and were described previously (VACV-GFP; ref. ⁵⁰) or were generated as described previously⁵¹. HSV-1 (KOS) and VACV were obtained from ATCC. HeLa CGAS-KO cells were treated with doxycycline for 4 days to induce cGAS, cGAS(NN) or cGAS(KK) expression, or HeLa cells were treated with doxycycline for 2 days to trigger enforced expression of SPSB3. Infections were performed by incubating virus inoculum (multiplicity of infection, 0.1) with cells for 3 h, before the cells were washed and cultured in complete DMEM (10% FBS, 1% penicillin–streptomycin–glutamine solution). After overnight incubation, cells were fixed and GFP⁺ cells were analysed by flow cytometry. The flow cytometry gating strategy is shown in Supplementary Fig. 2. To assess mRNA expression levels of IFNB1, cells were infected with HSV-1 (KOS) (multiplicity of infection, 0.1) or VACV (multiplicity of infection, 1) for 18 h.

Reporting summary

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