Telomeric repeat-containing RNA G-quadruplexes trigger ZBP1-mediated cell death

Domain: chemistry
Published: 2026-02-23
Editorial outcome: major_revision

Abstract
Z-DNA-binding protein 1 (ZBP1) is a nucleic acid sensor with two Z-DNA-binding domains (Zα domains) that bind Z-DNA and Z-RNA, mediating inflammatory cell death in development and disease. A recent study has demonstrated that the truncated isoform ZBP1-S senses telomeric repeat-containing RNA (TERRA), activating innate immune signaling and triggering cell death. However, the precise mechanism on how ZBP1-S senses TERRA remains unknown. G-quadruplexes (G4s) are non-canonical secondary structures of nucleic acids that can form within guanine (G)-rich strands of DNA or RNA, including TERRA. Here, we demonstrate that ZBP1-S directly senses G4 structures within TERRA, leading to ZBP1-S oligomerization and subsequent induction of inflammatory cell death. Furthermore, G4-specific binding ligands can compete with ZBP1-S for binding to TERRA G4s and inhibit ZBP1-S mediated activation of interferon signaling. Therefore, this work deciphers a hitherto uncharacterized mechanism that links TERRA G4s with ZBP1-S activity in triggering inflammatory cell death.

Introduction
DNA-binding protein 1 (ZBP1), initially identified as an interferon (IFN)-induced tumor-associated protein, possesses domains capable of binding to nucleic acids in the form of a left-handed helix (Z-form)1–3. ZBP1 has recently emerged as a crucial innate immune sensor that governs the activation of both programmed cell death and inflammation across various conditions, including cancer, virus infection, and embryonic development4. The ZBP1 gene encodes two Z-DNA-binding domains (Zα1 and Zα2 domains) at the N-terminal, which have been observed to bind to Z-DNA, B-DNA, and RNA5,6. Additionally, there are two homotypic interaction motifs (RHIM) domains present in the central region of ZBP1 that facilitate interactions with other proteins containing RHIM domains7,8. These RHIM domains play a crucial role in mediating cell death and inflammatory responses triggered by ZBP19,10. Furthermore, it has been reported that the conserved C-terminal domains of ZBP1 interact with TBK1 and IRF3 to induce type I interferon (IFN) responses upon exposure to immunostimulatory DNA4.

A recent study has demonstrated that dysfunctional telomeres trigger innate immune responses via mitochondrial TERRA-ZBP1 complexes to eliminate cells with a predisposition for neoplastic transformation11. TERRA interacts with ZBP1 to activate the innate immune adapter protein mitochondrial antiviral-signaling protein (MAVS), but the precise interaction mechanism of TERRA between ZBP1 is still unknown. Zα domain of ADAR1 has been shown to interact with MYC promoter G-quadruplex (G4) formed in NHEIII1 region and represses MYC transcription12. G4s are stable, non-canonical nucleic acids secondary structures adopted by guanine-rich DNAs or RNAs, which have been shown to be involved in modulating many processes of cellular biology and are reviewed as potential therapeutic targets for various human disease, including cancer, neurodegenerative disorder and virus infection13–18. Given that TERRA contains abundant G4 forming sequences, therefore, we speculated that TERRA interacts with ZBP1 via RNA G4 structures.

In this study, the interactions between the Zα domains of ZBP1 and TERRA G4 have been demonstrated. The truncated ZBP1 isoform (ZBP1-S) engages with TERRA G4 via Zα2. In addition, G/A mutant of TERRA impairs its interaction with ZBP1-S and inhibits ZBP1-S mediated MAVS-dependent interferon response and inflammatory cell death. Intriguingly, TERRA G4 oligonucleotides can activate ZBP1 and induce cell death, while G4-specific ligands pyridostatin (PDS) and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4) inhibit ZBP1-S mediated activation of innate immune signaling by blocking the interactions between TERRA and ZBP1-S. This work deciphers the mechanism on how ZBP1-S senses TERRA, indicating the key roles of G4 structures in ZBP1 mediated activation of innate immune signaling of inflammatory cell death.

Results
The Zα domains of ZBP1 interact with TERRA G4
Recent studies have demonstrated that TERRA serves as an immunostimulatory nucleic acid species recognized by ZBP111; however, the precise mechanism underlying how ZBP1 senses TERRA remains elusive. Alternative splicing of human ZBP1 generates two predominant isoforms: the full-length ZBP1 (ZBP1-L) harbors two Z-DNA-binding domains (Zα1 and Zα2), whereas the truncated isoform (ZBP1-S) lacks Zα1 but retains Zα2 (Supplementary Fig. 1a). Zα domains of ADAR1 have been shown to recognize G4 structure12, and TERRA can form RNA G4 structures19. We first investigated whether Zα1 and Zα2 domains of ZBP1 could interact with TERRA G4. To this end, we synthesized FAM-labeled 21 nucleotide (nt) TERRA G4 wild type RNA (TERRA-G4WT), its mutant RNA (TERRA-G4Mut, with G/A mutations), and Flag-fused Zα1 and Zα2 domain of ZBP1 (Supplementary Fig. 1b and Supplementary Data 1). By detection of the FAM fluorescence intensity following TERRA G4 pull-down assays via Anti-Flag Magnetic Beads (Fig. 1a), we found that both Zα1 and Zα2 domains can directly bind to TERRA-G4WT, but not TERRA-G4Mut (Fig. 1b). To explore the interaction between RNA G-quadruplex and ZBP1, one dimensional 1H-NMR titration experiments were pursued. As a control, TERRA G-quadruplex alone showed essentially 12 characteristic guanine imino proton peaks (partially overlapped) at 11.2-11.7 ppm, likely corresponding to an intramolecular G-quadruplex structure with three stacked G-tetrads (Supplementary Fig. 1c–e). Upon increasing the concentration of ZBP1, both the chemical shift perturbation and peak broadening became more and more evident among guanine imino proton peaks of the RNA G-quadruplex, revealing their interactions between TERRA G4 and ZBP1.

It is known that G-quadruplex can form more complex higher order G4s20, such as dimer (TERRA G4-2) (Supplementary Fig. 1f–h); therefore, we further investigated the interaction between Zα domains and the higher order TERRA G4s (Fig. 1c). As expected, we found that Zα domains can bind to higher order TERRA G4s (Supplementary Fig. 1i, j). Both Zα1 and Zα2 preferred to bind to more complex higher-order G4 with higher binding affinity compared to monomer G4 (Fig. 1d). Consistently, when incubating Zα domains with the mixture of FAM-labeled 21 nt TERRA G4 and G4-2, Zα domains preferred to capture higher-order TERRA G4-2 rather than 21 nt TERRA G4, indicating its higher binding ability for higher-order TERRA G4 (Supplementary Fig. 1k). In addition, the binding affinity of Zα2 to TERRA G4 was higher compared to that of Zα1 (Fig. 1e). We also further estimated the binding constant (Kd) of Zα1 and Zα2 for TERRA G4, respectively, and found that Zα1 and Zα2 indeed prefer higher-order TERRA G4 (Fig. 1f). Taken together, our results indicate that Zα1 and Zα2 domains of ZBP1 can interact with TERRA G4.

ZBP1-S engages with TERRA G4 via Zα domains
ZBP1-S has been demonstrated to sense TERRA, thereby activating innate immune signaling pathways essential for inflammatory cell death11. Given that ZBP1-S lacks Zα1 but retains Zα221, we hypothesized that ZBP1-S interacts with TERRA via Zα2 domain. First, to model the interaction of ZBP1-S with G-quadruplex, we utilized AlphaFold prediction1 on the second Z-DNA binding domain (PDB ID: 3EYI) in complex with a known human telomeric RNA G4 (PDB ID: 3IBK). The overall prediction has a high confidence (Fig. 2a), suggesting that the model is highly confident for the interaction between ZBP1 and G4, with the Zα interface supporting G4 binding. We also utilized Rosetta modeling to optimize the predicted complex, in which we observed the telomeric G4 followed a canonical RNA G4 pattern stabilized by hydrogen bonds between the guanine groups and further enhancement of the structural integrity through coordination with K+ at each layer (Fig. 2b). Moreover, the interaction between ZBP1-S and G4 was achieved through an intensive interaction network between the S8, Q9, and R52 residues (corresponding to S106, Q107 and R150 in ZBP1) that stabilized the outward-pointing residues in the G4 U7 and A8, thus stabilizing the quadruplex conformation (Fig. 2c). Other interactions also played roles in the formation of the complex such as the guanidinium group on R44 (corresponding to R142 in ZBP1) could interact with the backbone of the quadruplex on G3, thus enriching the stability of the ZBP1-G4 complex (Fig. 2d). Through the structure model, we contended that the interaction of the second domain of ZBP1 may play a crucial role in recognizing and stabilizing the G-quadruplex through the Zα interface. Then, to further substantiate this hypothesis, we did disrupt the TERRA G4-binding by point mutations (S106A, Q107A, R142A and R150A, ZBP1-S mZα2)5 (Supplementary Fig. 2a). By detection of the FAM fluorescence intensity following TERRA G4 pull-down assays, we found that ZBP1-SWT (ZBP1-S) can bind to TERRA-G4WT, but not TERRA-G4Mut (Fig. 2e–g and Supplementary Fig. 2b). Consistently, ZBP1-S also preferred to capture higher-order TERRA G4 (Fig. 2g), and we determined the Kd of ZBP1-S for TERRA G4 under our experimental conditions: Kd [TERRA G4]=48.82 ± 0.98 nM and Kd [TERRA G4-2]=22.30 ± 0.87 nM (Fig. 2h). As expected, electrophoresis mobility shift assay (EMSA) and TERRA G4 pull-down assays indicated that Zα2 mutant impaired the binding of ZBP1-S to TERRA G4 (Fig. 2j and Supplementary Fig. 2c, d). And a supershift assay using a validated anti-ZBP1 antibody confirmed the formation of ZBP1/TERRA G4 complexes (Fig. 2k). Consistently, a pulldown assay employing biotinylated TERRA G4 RNA verified the interaction between TERRA G4 and ZBP1 in living cells (Supplementary Fig. 2e). To further explore the interaction between ZBP1-S and TERRA G4, we synthesized FAM-labeled TERRA G4-2 (G4-2-WT) and its G/A mutant (G4-2-Mut), followed by transfection into HEK293T cells along with Flag-fused ZBP1-S. Our results demonstrated that the yellow regions in the merged image indicated a high degree of co-localization between G4-2-WT and ZBP1-S, whereas G4-2-Mut and ZBP1-S exhibited minimal co-localization (Fig. 2l and Supplementary Fig. 2f), suggesting the ZBP1-S interacted with TERRA G4 in living cells. Worth to note, in addition to the key residues (S106, Q107, R142, and R150), mutations in the canonical Z-DNA/Z-RNA binding residues (N141 and Y145), as previously reported11, similarly abolish TERRA G4 binding (Supplementary Fig. 2g–i). Collectively, our results indicate that ZBP1-S engages with TERRA G4 via Zα2 domain.

TERRA G4 promotes ZBP1 oligomerization and cell death
We next investigate whether TERRA G4 is essential for ZBP1 to activate innate immune signaling and induce inflammatory cell death. First, we investigated whether endogenous TERRA can form G4 structures in living cells. RNA immunoprecipitation (RIP) assays showed the enrichment of TERRA in complexes precipitated by G4-specific antibody BG4 as compared with control IgG (Supplementary Fig. 3a), indicating the G4 formation within endogenous TERRA. In addition, we conducted the TERRA (TERRA WT) and its G/A mutant (TERRA Mut) expression vectors for expressing TERRA and its mutant in living cells. To generate full-length TERRA transcripts, we cloned the human chromosome 15q subtelomeric regions (As a representative) containing the TERRA start sites before the TTAGGG or TTAGAG repeats (Fig. 3a, b). Upon binding to its nucleic acid substrates, ZBP1 can undergo oligomerization, thereby initiating innate immune signaling or triggering cell death3,11. Thus, we first explored whether TERRA G4 was essential for ZBP1-S oligomerization. By performing bimolecular fluorescence complementation (BiFC) assays in HEK293T cells, we observed a significant induction of the BiFC signal upon overexpression of TERRA WT, whereas no such effect was observed with TERRA (Fig. 3c, d). Consistently, by performing semi-denaturing detergent agarose gel electrophoresis (SDD-AGE), we found that TERRA WT could induce ZBP1-S oligomerization in the ZBP1-S overexpressed MCF-7 cells, but not TERRA (Fig. 3e). In addition, to directly visualize ZBP1-S oligomerization in living cells, we performed Airyscan super-resolution microscopy. As shown in Fig. 3f, ZBP1-S formed distinct oligomeric filaments upon induction of TERRA G4s (Fig. 3f), consistent with previous study11.

To investigate the effects of TERRA G4 on ZBP1-S mediated cell death, we constructed ZBP1-knockout MCF-7 cells for reconstituting with ZBP1-S (Supplementary Fig. 3b). We found that ZBP1-S overexpression hardly affect the cell viability (Supplementary Fig. 3c, d). However, ZBP1-S significantly induced cell death in TERRA WT overexpressed MCF-7 cells, but not in TERRA overexpressed cells (Fig. 3g–i). In addition, ZBP1-S co-expressed with TERRA WT could activate interferon (IFN) signaling, marked by TBK1, IRF3, and STAT1 phosphorylation (Supplementary Fig. 3e). IFN-stimulated genes (ISGs) (such as IFNB1, CCL5, CXCL10, and ISG15) were induced by ZBP1-S in TERRA WT overexpressed cells (Fig. 3j). Previous studies have demonstrated that ZBP1-S mediated innate immune signaling induced by TERRA is mediated by MAVS11. Indeed, we found that MAVS knockdown blocked ZBP1-S mediated up-regulation of ISGs in TERRA WT overexpressed cells (Fig. 3k and Supplementary Fig. 3f). Finally, ZBP1-knockout cells were reconstituted with mutant forms of ZBP1-S (Supplementary Fig. 4a, b) and tested for their susceptibility to induce ISGs and undergo cell death. We found that cells reconstituted with ZBP1-S containing mutations in key conserved residues involved in nucleic acid binding or RHIM1-based interaction (I206A, Q207A, I208A, G209A)7,22 did not induce ISGs and cell death in TERRA WT overexpressed cells (Supplementary Fig. 4c, d), In contrast, the cells reconstituted with ZBP1-S containing mutations in RHIM2 (V264A, Q265A, L266A, G267A) could still induce ISGs and cell death in TERRA WT overexpressed cells, indicating that RHIM2 is largely dispensable for this function (Supplementary Fig. 4c, d). And the same results were obtained in MDA-MB-231, Hela and A549 cells (Supplementary Fig. 5a–g). Worth to note, TERRA with distinct subtelomere regions (Human chromosome 9p, Xp, and Xq) or TERRA G4 oligonucleotides had the ability to activate innate immune signaling and elicit cell death (Supplementary Figs. 6a–c and 7a–c), thereby emphasizing the pivotal roles of G4 structure for recognizing TERRA by ZBP1-S. Taken together, our results indicate that TERRA G4 facilitates ZBP1-S oligomerization through its Zα2 domain, thereby promoting inflammatory cell death.

G4-specific ligands inhibit ZBP1-mediated activation of innate immune signaling
G4-specific ligands have been developed and successfully used as tools for exploring the intricate biological functions and mechanisms mediated by G4, as well as potential therapeutic agents targeting human diseases23–26. Many G4-specific ligands have been demonstrated to recognize and stabilize telomeric DNA G4 and TERRA G419. G4-specific ligands PDS and TMPyP4 (Fig. 4a) have been used to target DNA and RNA G4s25. Both PDS and TMPyP4 can recognize TERRA G427,28. Thus, we next investigated the effects of PDS and TMPyP4 on the interaction between TERRA and ZBP1-S, as well as ZBP1-S-mediated activation of innate immune signaling. First, we found that both PDS and TMPyP4 were competitive with the Zα domains for binding to TERRA G4 (Fig. 4b and Supplementary Fig. 8a, b), showing that PDS and TMPyP4 could inhibit the binding of ZPB1-S to TERRA G4 (Fig. 4c and Supplementary Fig. 8c). BiFC assays showed that PDS and TMPyP4 inhibited ZBP1 oligomerization (Fig. 4d, e). Furthermore, PDS and TMPyP4 treatment significantly decreased the expression of ISGs in the ZBP1-S and TERRA WT overexpressed cells (Fig. 4f). However, we found that the ability to interfere with ZBP1 recognition is not a universal property of G4 ligands. The G4 ligand BMVC inhibited the interaction between ZBP1 and TERRA G4 with comparable efficacy to PDS and TMPyP4 under our experimental conditions, but PhpC or Phen-DC3 just showed slightly inhibition effect on ZBP1 binding to TERRA G4 (Supplementary Fig. 8d–g), indicating that the ability of G4 ligands to interfere with the ZBP1-TERRA G4 interaction is influenced by their binding mode, preferred G4 topology, or specific binding site within the TERRA G4. In addition, we investigated whether RNA helicases such as DHX36 could influence ZBP1-mediated activation of innate immune signaling by unwinding TERRA G4s. As expected, DHX36 knockdown significantly increases the expression of ISGs in cells co-overexpressing ZBP1-S and TERRA (Supplementary Fig. 9a, b), indicating further activation of ZBP1-mediated innate immune activation. These results suggest that DHX36 acts as a negative regulator of this pathway. We proposed a model wherein DHX36 normally functions to unwind or remodel TERRA G4s, thereby dismantling the activating ligand for ZBP1 and suppressing signaling.

Inspired by the telomeric RNA G4, we next explored the potential interaction between the Zα domains of ZBP1 and other cellular RNA G4s. We chose two well-characterized RNA G4s: NRAS and BCL-2 mRNA 5’ untranslated region (5’UTR) G4s29,30 for subsequent study. We found that Zα domains can interact with these two RNA G4s (Supplementary Fig. 10a, b). Consistently, both ZBP1-L and ZBP1-S could also bind to these two RNA G4s (Supplementary Fig. 10c, d), implying the potential of ZBP1 in recognizing other RNA G4s besides TERRA G4. Moreover, we investigated the ability of Zα domains to bind various types of nucleic acids, including G4 DNA, ssRNA, dsRNA, ssDNA, and dsDNA. Our results demonstrate that Zα domains exhibit binding affinity toward G4 DNA structures, such as Myc G4 and KRAS G4, but do not bind ssRNA, dsRNA, ssDNA, or dsDNA (Supplementary Fig. 11a–d) under our experimental conditions. The biological function of ZBP1 in sensing other cellular DNAs and RNAs with G4 structures warrants further investigation in future studies.

Methods
Oligonucleotides and chemical compounds
All oligonucleotides used in this study were synthesized by Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China). Chemical compounds were purchased from MedChemExpress (MCE, USA) and used without further purification.

TERRA G4 pull-down assays
Two μM FAM-labeled TERRA G4 RNA and flag-fused Zα1 and Zα2 domains of ZBP1 or ZBP1 proteins, as indicated concentrations, were mixed well in test tube, and then the Anti-Flag Magnetic Beads were added to pull down G4/protein complexes. After rinsing and dissolving, the pull-down FAM-G4 was detected by fluorescence spectrophotometer (JASCO FP-6500). For the biotinylated TERRA G4 pull-down assay, biotinylated TERRA G4 was transfected into MCF-7 cells, followed by cell lysis and protein extraction. Streptavidin magnetic beads were then added to isolate G4/protein complexes, which were subsequently analyzed by western blotting.

AlphaFold prediction and structural analysis
The complex prediction was achieved in AlphaFold340, using the available ZBP1 second Z-DNA binding domain (PDB ID: 3EYI) and human telomeric RNA G-quadruplex (PDB ID: 3IBK) from the available structure in the protein data bank5,41. The generated structure was optimized through PyRosetta42 under the FastRelax protocol to perform energy minimization. Further analysis was conducted in PyMol (The PyMOL Molecular Graphics System, Version 3.0, Schrödinger, LLC).

Recombinant protein expression and purification
To express the recombinant protein, the pET-28a ZBP1-L and ZBP1-S vectors were introduced into the Escherichia coli BL21 (DE3) strain. The bacteria were cultured in Luria-Bertani (LB) broth supplemented with 50 μg/ml kanamycin at 37°C until the optical density at 600 nm (OD600) reached approximately 0.6. The cultures were then cooled to 16°C, and expression was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for a duration of 16 h. Following induction, cell pellets were harvested via centrifugation and resuspended in chilled lysis buffer composed of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM DTT, and 1 mM PMSF. The cells were lysed using sonication for 30 min, alternating between 5 s of pulses and 25 s of rest, all while maintaining the temperature with ice. The lysate was subsequently clarified by centrifugation at 5180 × g for 2 h. The hexahistidine-tagged fusion proteins from the supernatant were purified using a HisTrap FF prepacked column (#17531901; Cytiva). The proteins were further desalted using a HiTrap Desalting prepacked column (#29048684; Cytiva). The recombinant proteins were stored in −80°C. For the expression of flag-fused Z-DNA-binding domains (Zα1 and Zα2 domains), we used TNT® Quick Coupled Transcription/Translation System (Promega) according to the manufacturer’s protocol.

Nuclear magnetic resonance (NMR) spectroscopy experiments
One dimensional NMR data were collected at 298 K on a 500 MHz Bruker spectrometer with a cryoprobe. The gradient-tailored excitation (WATERGATE) was used to suppress the water signal for 90% H2O/10% D2O samples. The stock solution of G-rich TERRA RNA oligonucleotide (AGGGUUAGGGUUAGGGUUAGGG) was first dissolved in 5 mM potassium phosphate buffer (K-Pi, pH 6.8). Next, this RNA sample was heated at 95°C for 5 min and then slowly cooled to room temperature to ensure the complete formation of a G4 structure. The stock solution of protein was initially dissolved in 1× PBS buffer. For NMR titration experiments, 0.1 mM RNA TERRA G4 sample was titrated stepwise with increasing amounts of protein, whereas the 0.1 mM RNA G4, as a reference, was only treated with corresponding amount of PBS alone without protein. All of the NMR titration spectra in Supplementary Fig. 2e, f were collected in a final buffer solution containing 3.6 mM K-Pi and 0.27× PBS. All data were processed and analyzed with Bruker Topspin 3.6.2 software.

Enzyme-linked immunosorbent assay (ELISA)
ELISAs were conducted to assess the binding affinity. Briefly, for investigating the binding constant (Kd) of Zα domains and ZBP1 with G4s, biotinylated G4 oligonucleotides were immobilized onto streptavidin-coated plates. And then the plates underwent incubation with Flag-tagged Zα domains and ZBP1 and HRP-conjugated anti-Flag antibody (86861S; Cell Signaling Technology, USA). Finally, the plates were incubated with TMB (3,3’,5,5’-tetramethylbenzidine, HRP substrate), and signal intensity was measured at 450 nm. The oligonucleotides are provided in Supplementary Data 1.

Cell culture
The HEK293T, Hela and A549 cell lines (obtained from the China Center for Type Culture Collection, CCTCC, Wuhan, China), as well as the MCF-7 and MDA-MB-231 cells (obtained from CCTCC, Wuhan, China) derived from human breast cancer, were cultured in a high-glucose medium known as Dulbecco’s modified Eagle’s medium (DMEM; HyClone, USA). This medium was supplemented with 10% fetal bovine serum (FBS; HyClone, USA), along with 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were incubated at a temperature of 37°C in a humidified environment containing 5% CO2.

Transfection of plasmids
The transfection of plasmids was performed using Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer’s instructions. Briefly, cells were seeded until they reached a confluency of 70–90% prior to transfection. The plasmids and Lipofectamine 2000 reagent were combined in serum-free DMEM medium. After incubating the mixtures at room temperature for 15 min, they were added to the cells in each dish. The collected cells at specified time points corresponded to those that had undergone transfection.

Viral packaging and infection
The vectors of interest, along with viral packaging plasmids (pMD2.G and psPAX2), were introduced into HEK293T cells using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s guidelines. Briefly, cells were seeded in 60 mm culture dishes at a confluency ranging from 70% to 90% during transfection. A mixture comprising 6 µg of target plasmids, 2 µg of pMD2.G (Addgene #12259), 4 µg of psPAX2 (Addgene #12260), and 30 µL of Lipofectamine 2000 reagent was prepared in serum-free DMEM. After incubating the mixtures for a duration of 20 min at room temperature, they were added to the cells in each dish. The media was replaced after 6 h post-transfection. Virus supernatant was collected after 48 h, filtered through a filter with pore size measuring at 0.45 μM, and stored at −80°C until further use. For infection experiments, virus supernatant was incubated with target cells for an additional period lasting 48 h at a dilution ratio equivalent to 1:10 with polybrene (8 μg/mL). Subsequently, infected cells were digested and counted before being reseeded sparsely in new culture dishes supplemented with selection agents administered after 6 h.

Western blotting
To analyze the proteins in whole-cell lysates, we used RIPA buffer (CWBIO, Beijing, China) to lyse the cells. The total protein samples (10–20 μg) were then suspended in Laemmli buffer (63 mM Tris-HCl, 10% glycerol, 2% SDS, 0.0025% bromophenol blue, pH 6.8) and subjected to electrophoresis on SDS-polyacrylamide gels. Subsequently, the proteins were transferred onto a polyvinylidene difluoride membrane. For antibody detection of specific proteins such as Phospho-IRF3 (Ser396) (AF5838; Beyotime, Shanghai, China), Phospho-STAT1 (Tyr701) (AF5935; Beyotime, Shanghai, China), Phospho-TBK1 (Ser172) (AF595959; Beyotime, Shanghai, China), Flag (TA-05, ZSGB-BIO, China), ZBP1 (ab232977; Abcam, USA), or GAPDH (CW0098M; CWBIO Beijing, China), we performed an incubation step followed by incubation with secondary antibodies conjugated to horseradish peroxidase (HRP). Anti-mouse antibody or anti-rabbit antibody from Beyotime (Shanghai, China) was used as secondary antibody. To visualize the immunoreactive bands on the membrane surface after incubation with HRP-conjugated antibodies, washing steps were completed using SuperSignal™ West Pico chemiluminescent substrate kit from Thermo Fisher Scientific (Waltham, MA, USA). Finally, the Western blotting detection system from Bio Rad (Hercules, CA, USA) was employed for band visualization. In order to ensure accurate quantification of protein levels during western blotting assays, GAPDH was utilized as a loading control.

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE)
Cells were lysed by lysis buffer and loaded onto 1.5% agarose gel. After electrophoresis in running buffer (1× TBE, 0.1% SDS) for 2 h with a constant voltage of 50 V at 4°C, proteins were transferred to PVDF membranes with a Wet/Tank transfer blotting system (Bio-Rad Laboratories) for western blotting analysis.

Nondenaturing polyacrylamide gel electrophoresis
Native gel electrophoresis was carried out on acrylamide gel (15%) and run at 25°C, 1× TB buffer containing 10 mM KCl, and was silver stained. To prepare the final loading sample, ZBP1-S (2 μM or 4 μM) or ZBP1-S (2 μM or 4 μM) proteins were incubated with annealed TERRA G4 WT (1 μM) or TERRA G4 Mut (1 μM), respectively, at 4°C for 12 h. For the supershift assay, ZBP1-S (4 μM) or ZBP1-S (4 μM) proteins and ZBP1 antibody (2 μM; ab232977; Abcam, USA) were added to the annealed TERRA G4 samples (1 μM) and incubated at 4°C for 12 h. In competition assays involving unlabeled TERRA G4, annealed FAM-labeled TERRA G4 (1 μM) was incubated with ZBP1-S (4 μM) protein in the presence of annealed unlabeled TERRA G4 (1 μM or 2 μM) at 4°C for 12 h.

Immunofluorescence (IF)
Cells were washed twice in 1× PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100-PBS for 15 min, and then blocked for 30 min with 3% BSA-0.2% Triton X-100-PBS. Furthermore, cells were incubated with primary Mouse Flag (TA-05, ZSGB-BIO, China) for 16 h at 4°C. Secondary antibody was Rhodamine (TRITC) Goat Anti-Mouse IgG (H+L) (AS026; ABclonal, China) used in a 1:1000 dilution for 2 h at 37°C. DAPI (P0131; Beyotime, Shanghai, China) was added for 2 min in the dark before taken images with confocal laser-scanning microscope LSM 700 (Carl Zeiss, Germany). The fluorescence value of EGPF was detected by IF assays. For image acquisition, all confocal microscopy images were acquired under identical laser power, gain, and exposure settings within the same experiment. For experiments quantifying the colocalization foci between ZBP1-S and FAM-labeled TERRA G4 was strictly defined not by simple channel overlap, but by the presence of a probe focus as defined above, that spatially coincided with a ZBP1-S punctum. A foci was defined as a discrete, punctate signal cluster meeting the following criteria: its mean pixel intensity exceeded a threshold set at three standard deviations above the mean background intensity of a cell-free region in the same image; its area, after automated particle detection, was constrained between 0.1 μm² and 1.0 μm² to exclude diffuse signals and large, non-specific aggregates. For colocalization analysis, spatial overlap was quantified by registering a colocalization event only when ≥60% of the pixels within a probe focus overlapped with the ZBP1-S mask. The results are presented as the number of colocalization events per cell. For ZBP1-S filament visualization and quantification, Zeiss Confocal LSM980+Airyscan 2 was used. ZBP1-S filaments in Airyscan images were defined as elongated structures meeting objective morphological criteria: a skeletonized length >0.5 µm, a length-to-width ratio >3, and a form factor <0.5. Images were processed in ImageJ: after background subtraction and thresholding, filaments were enhanced using a directional filter, skeletonized, and analyzed with the “Analyze Skeleton” plugin. The number of filaments per cell was automatically quantified.

Bimolecular fluorescence complementation (BiFC)
BiFC assays were performed to investigate ZBP1 oligomerization. Briefly, the gateway cloning technique was utilized to introduce the ZBP1 gene into GFP10C and GFP11C vectors, resulting in the creation of two ZBP1 constructs containing complementary fragments of GFP. These constructs were co-transfected with TERRA and ZBP1-S into cells, allowing for the assessment of ZBP1 oligomerization based on the reassembly of split GFP fragments. Subsequently, cells were fixed using 4% PFA and counterstained with DAPI in preparation for imaging.

Quantitative real-time polymerase chain reaction (qRT-PCR)
The cultured cells were subjected to RNA extraction using the RNApure tissue & cell kit (CW0560; CWBIO, China) following the manufacturer’s instructions. To synthesize cDNAs, 500 ng of total RNAs were utilized with the PrimeScript TM RT Master Mix (RR036A; Takara, Japan) according to the manufacturer’s protocol. The qRT-PCR assays were conducted on a qTOWER 2.0 quantitative real-time PCR system (Analytik Jena, Germany) using BeyoFastTM SYBR Green qPCR Mix (D7260; Beyotime, China). The comparative Ct method was employed to calculate the relative expression levels of RNAs. Additionally, melting curve analyses were performed for all PCRs in order to eliminate any non-specific amplification concerns. The primers were described in Supplementary Data 1.

RNA immunoprecipitation (RIP)
RIP assays were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore, USA) following the manufacturer’s instructions. G4 specific antibody BG4 was used for the RIP. Briefly, Cells cultured in 10-cm plates were washed twice with ice-cold PBS, scraped, and pelleted by centrifugation. The cell pellet was resuspended in complete RIP lysis buffer. Lysate was incubated with antibody-conjugated beads overnight at 4°C in immunoprecipitation buffer. Beads were collected magnetically and washed five times with RIP wash buffer. Immunoprecipitated RNA was eluted by incubation with proteinase K buffer at 55°C for 30 min, followed by phenol:chloroform extraction and ethanol precipitation. Purified RNA was resuspended in RNase-free water and analyzed by qRT-PCR.

Cell viability and cell growth assays
Cell viability and cell growth of MCF-7 and MDA-MB-231 cells with Bio-PDC treatment were assessed using Cell Counting Kit-8 (BA00208; Bioss, China) according to the manufacturer’s protocol.

Statistical analysis
The data presented in the figures represent a minimum of three independent experiments. Each corresponding figure and its legend provide information on the number of samples per experiment, as well as details regarding statistical methods and reproducibility. Statistical analyses were conducted using SPSS 13.0 software (Chicago, IL, USA) and GraphPad Prism software (La Jolla, CA, USA). A Student’s t-test was employed to compare quantitative data between two groups. Significance was determined at P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Qin and colleagues demonstrate that the TERRA G-quadruplex (G4) directly binds ZBP1 and activates it to promote\ninflammation and programmed cell death. However, the reported findings lack substantial novelty, and the authors'\nconclusions are poorly supported by their weak experimental evidence.\nSeveral critical issues require clarification:\n1.Given prior studies showing that the Zα domain of ADAR1 interacts with DNA G-quadruplexes (G4) (PMID: 24813121),\nand considering the high structural similarity between the Zα domains of ZBP1 and ADAR1, the discovery that TERRA G4\nbinds ZBP1 offers little conceptual advancement. Moreover, a recent preprint (https://doi.org/10.1101/2024.06.29.601251) \nreports that RNAs with G-quadruplexes (rG4) bind both ADAR1 p150 and ZBP1 via their Zα domains, further undermining\nthe novelty of this manuscript.\n2.Weak Experimental Support for Key Claims\n1）Fig. 2f: The interaction between TERRA G4 and ZBP1-S appears extremely weak.\n2）Fig. 2i: The representative images show no clear difference in colocalization between G4 WT and G4-mut with ZBP1.\n3.The study relies entirely on synthetic nucleic acids, overexpression, and transfection assays, which are highly artificial.\nThere is no evidence that endogenous G4 structures adopt a Z-form conformation and activate ZBP1 under physiological\nconditions.\n4.Fig. 3d & 3f: The reported phenotypes are barely detectable, making it difficult to support the authors’ conclusions.\n5.Fig. 3 (ISG induction): The fold changes in ISGs are minimal, suggesting that TERRA G4 has only a marginal effect on\nZBP1 activation.\n6.While Figures 2C-D suggest novel binding sites through structural modeling, the authors did not experimentally validate\nthese predictions. Instead, they proceeded with Zα binding site mutants, a logical leap that lacks sufficient mechanistic\nsupport. Key concerns include: the assumed (but unproven) similarity between TERRA G4 and Z-form DNA. The failure to\nfollow up on predicted critical residues (e.g., N141, Y145) implicated by their own structural data.\n7.Several experiments lack essential controls. For example, Extended Data Fig. 2C-D omits a direct comparison between\nZBP1-S WT and Mut, which is crucial for interpreting functional differences.\n8.The study reports that ZBP1-S RHIM2 is necessary for inflammation and cell death (Extended Data Fig. 4, 5E),\ncontradicting prior work suggesting RHIM2 is largely dispensable. Whether this discrepancy reflects a context-dependent\nphenomenon or methodological differences remains unclear.", "reviewer_2": "The study by Qin et al. provides important mechanistic insight into how the short, human-specific isoform of ZBP1 (ZBP1-S)\nrecognizes telomeric repeat-containing RNA (TERRA) and activates MAVS-dependent antiviral signaling. ZBP1 is known to\ncontain tandem Zα domains that bind left-handed Z-DNA and Z-RNA; however, it has remained unclear how these domains\ncould recognize single-stranded RNAs such as TERRA. Building on prior work by Nassour et al. (Nature, 2023), which\ndemonstrated that ZBP1-S senses TERRA and triggers interferon (IFN) signaling and cell death during telomere crisis, this\nstudy reveals that TERRA adopts G-quadruplex (G4) RNA structures that are directly bound by the Zα2 domain of ZBP1-S. This\ninteraction is shown to be both necessary and sufficient to drive downstream immune signaling. Notably, this is the first\ndemonstration of a Zα domain directly engaging an RNA G-quadruplex, thereby expanding the known ligand repertoire of Zα\ndomains and identifying G4 RNA as a novel class of immunostimulatory structures. While previous studies have shown that\nZBP1 can be activated by viral infections and endogenous retroelements (EREs) through recognition of Z-form nucleic acids\n—including Z-RNAs produced during influenza, vaccinia virus infection, and retrotransposon expression—this work raises\nthe intriguing possibility that structured viral RNAs forming G4 motifs may also serve as ligands for ZBP1-S. However, to\nmove beyond a conceptual extension of the previously defined TERRA–ZBP1 axis, the study must more thoroughly define\nthe molecular basis of this interaction—particularly the structural diversity of G4 RNAs involved, whether multimeric or\nstacked G4 assemblies are required to promote ZBP1 multimerization and filament formation, and how ZBP1\noligomerization is initiated, stabilized, and spatially organized on these RNA scaffolds. In addition, the specificity of ZBP1-S\nfor G4 RNAs, as compared to other structured or unstructured RNA species, remains to be determined. It also remains to be\nclarified whether ZBP1 assembles into filament-like oligomers along RNA templates, similar to the cooperative filament\nformation observed for MDA5 during sensing of long dsRNA. Without addressing these mechanistic questions, the study\nrisks being interpreted as a refinement of earlier findings rather than a significant conceptual advance. Nevertheless, the\nidentification of RNA G-quadruplexes as functional ligands for ZBP1-S represents a novel and biologically meaningful\ndevelopment in the field of RNA structure–driven innate immunity, and clearly merits deeper investigation.\nMajor Comments\n1.Validation of ZBP1–TERRA G4 binding\nThe interaction between ZBP1-S and TERRA G4 RNA lies at the core of the manuscript’s mechanistic claims. While the\nEMSA results suggest a Zα2-dependent interaction, several technical issues limit interpretation. The free RNA band is\nheavily overexposed, and shifted complexes are faint and poorly resolved—especially at lower ZBP1 concentrations.\nFurthermore, minimal reduction in free RNA suggests weak binding efficiency or suboptimal assay conditions. I recommend\nrepeating the EMSA with optimized RNA:protein ratios and improved imaging for better contrast and resolution. A supershift\nassay using a validated anti-ZBP1 antibody (e.g., CST #60968) would help confirm complex identity. Inclusion of non-G4\nRNA controls and competition EMSAs with unlabeled TERRA would also strengthen specificity claims. Finally, a pulldown\nassay using biotinylated G4 RNA with ZBP1 Western blot detection would provide orthogonal confirmation.\n2.Evidence for ZBP1 oligomerization\nThe claim that ZBP1-S forms filament-like assemblies is currently based on immunofluorescence, but the resolution is\ninsufficient to distinguish true filaments from puncta or aggregates. Some figures even suggest primarily punctate\nlocalization with TERRA. A plausible model is that ZBP1 first binds TERRA G4 RNA to form puncta, and then oligomerizes\ninto filaments along the RNA scaffold, as seen with MDA5 and other sensors. This hypothesis should be explicitly tested\nand depicted. Higher-resolution microscopy (e.g., SIM, Airyscan, STED) would help clarify morphology. Biochemical\nvalidation is also necessary: native or blue native PAGE, crosslinking, or SEC-MALS could reveal oligomerization states.\nReconstitution with synthetic G4s could test whether multimeric G4 architecture promotes ZBP1 filament formation. If ZBP1\ndoes form extended assemblies, cryo-EM or negative-stain EM would provide valuable structural insight.\n3.Broader characterization of Zα domain ligand specificity\nGiven that ADAR1’s Zα domain can bind G4 DNA, it would be highly informative to test whether ZBP1’s Zα domains can\nalso bind structured DNA—including G4 DNA—or other nucleic acid types. Comparative binding across ssRNA, dsRNA,\nssDNA, dsDNA, RNA:DNA hybrids, and G4 DNA would clarify whether ZBP1 is emerging as a general nucleic acid sensor.\nThis would significantly expand the functional understanding of ZBP1-S and help position it within the broader landscape\nof innate immune pattern recognition receptors.\n4.Mechanistic comparison with RIG-I and MDA5\nThe authors should more explicitly address whether ZBP1 follows a mechanism more analogous to RIG-I (short dsRNA,\npolyubiquitin-assisted tetramerization) or MDA5 (long dsRNA-guided filament formation). This comparison would help\ncontextualize the findings and clarify whether ZBP1 represents a distinct G4-based sensing pathway.\n5.Role of multimeric G4 structures\nRNA G4s exhibit diverse topologies, including monomeric, stacked, and intermolecular forms. The manuscript would benefit\nfrom deeper biophysical characterization of the TERRA G4s used—via CD spectroscopy, native PAGE, or ThT/NMM\nfluorescence. Comparative assays with synthetic G4s of defined architecture (e.g., dimers, trimers, loop-length variants)\ncould test whether ZBP1 distinguishes between them. This would clarify whether G4 multimerization state influences ZBP1\nactivation or oligomerization.\nAdditional Comments\n•Split-GFP assay: The split-GFP results for ZBP1-S oligomerization are ambiguous. The GFP signal appears diffusely\nnuclear and cytoplasmic, rather than as discrete puncta or filaments. This undermines the conclusion that ZBP1\noligomerizes in a template-dependent manner. The authors should clarify this interpretation, possibly using higher-resolution\nimaging or quantification.\n•ZBP1 knockout validation: The use of ZBP1-KO MCF-7 cells is a key experimental component, but no data are shown to\nconfirm knockout efficiency. At minimum, a Western blot demonstrating loss of endogenous ZBP1 is required.\n•Figure legends and replicates: Several figure legends lack clarity, and the number of biological replicates is not\nconsistently reported. Please revise legends to include key experimental details, number of replicates, and statistical\nanalyses.\nSummary\nThis study addresses a biologically compelling and timely question by investigating how the human-specific ZBP1-S\nisoform senses structured RNA to activate innate immunity. While ZBP1 has been extensively studied in the context of\nnecroptosis in mice, its activation mechanisms in humans remain underexplored. The identification of TERRA G4 RNA as a\nfunctional ligand for ZBP1-S via a Zα domain is a notable conceptual advance. However, to fully realize the impact of this\ndiscovery, additional clarification is needed on (1) the structural nature and specificity of the ZBP1–TERRA G4 interaction,\nand (2) biochemical and structural evidence for ZBP1 oligomerization. The authors are well-positioned to address these\npoints, and doing so would significantly strengthen the manuscript and offer foundational insights into a novel human innate\nimmune mechanism.", "reviewer_3": "In the submitted manuscript, Qin et al. reported that ZBP1-S interacts with TERRA G-quadruplexes (G4s) through its Z-DNA-\nbinding domains (Zα). TERRA G4 structures are critical for ZBP1-S-mediated, MAVS-dependent interferon responses and\ninflammatory cell death. G4-specific ligands (PDS and TMPyP4) competitively inhibit ZBP1-S binding to TERRA G4s,\nthereby suppressing ZBP1-S-mediated activation of interferon signaling. In contrast, TERRA G4 oligonucleotides can\ninduce ZBP1-S-mediated inflammatory cell death. These findings elucidate the precise mechanism by which ZBP1-S\nrecognizes TERRA, highlighting the essential role of G4 structures in ZBP1-mediated activation of innate immune signaling\nand inflammatory cell death.\nAfter reading this work, my feeling is taht the work is novel and important to understanding G4 biology and ZBP1 mediated\ninnate immune signaling and inflammatory cell death. The authors have employed appropriate experimental controls and\napplied suitable statistical analyses to support their key conclusions, thus I am pleased to recommend the acceptance of this\nmanuscript for publication in Nature Communications. The major and minor comments of this work were outlined below,\nwhich I believe these would further enhance the clarity and impact of the manuscript.\nMajor points:\n1.The author demonstrated that ZBP1-S recognizes TERRA G4 structures, thereby activating ZBP1-S- and MAVS-\ndependent interferon responses and inflammatory cell death. It is known that the recognition of endogenous Z-form nucleic\nacids by ZBP1 initiates RIPK3-dependent necroptosis and inflammation. This raises the question: does the sensing of\nendogenous G4 structures by ZBP1 also induce RIPK3-dependent necroptosis and inflammation?\n2.Only two proteins containing Zα domains have been identified in mammals: ADAR1 and ZBP1. A pertinent question\narises: does ADAR1 recognize TERRA G-quadruplex (G4) structures? If so, what is the functional relationship between\nZBP1, ADAR1, and TERRA G4?\n3.In Figure 4 and Extended Data Figure 8, the author demonstrated that PDS and TMPyP4 inhibit ZBP1-mediated\nactivation of innate immune signaling by blocking the interaction between Zα domains and TERRA G-quadruplex (G4)\nstructures. How do G4 ligands interfere with the interaction between Zα domains and TERRA G4? Do these ligands\ncompete with TERRA G4 for binding to Zα domains?\nMinor points:\n1.In Fig 2i, I believe it should be “FAM-G4-2” rather than “FAM-G4-4”?\n2.The author demonstrated that PDS and TMPyP4 block the interaction between Zα domains and TERRA G4 structures.\nCan all G4 ligands exert such an effect? The author is encouraged to validate the effect using several additional G4 ligands.\n3.Do RNA helicases, such as DHX36, influence ZBP1-mediated activation of innate immune signaling through the\nunwinding of TERRA G4 structures?\n4.The quality of the Western blot results presented in Extended Data Figure 3d requires improvement."}, "author_response": "Next is the summarization of our point-by-point response to the comments raised by\nthe reviewers.\nReviewer #1:\nQin and colleagues demonstrate that the TERRA G-quadruplex (G4) directly\nbinds ZBP1 and activates it to promote inflammation and programmed cell death.\nHowever, the reported findings lack substantial novelty, and the authors' conclusions\nare poorly supported by their weak experimental evidence.\nSeveral critical issues require clarification:\nResponse:\nThank you for reviewing our manuscript and your insightful comments. The\nsuggestions and inputs are helpful and have been implemented in the manuscript.\nPlease see our point-to-point response below.\n1.Given prior studies showing that the Zα domain of ADAR1 interacts with DNA\nG-quadruplexes (G4) (PMID: 24813121), and considering the high structural\nsimilarity between the Zα domains of ZBP1 and ADAR1, the discovery that TERRA\nG4 binds ZBP1 offers little conceptual advancement. Moreover, a recent preprint\n(https://doi.org/10.1101/2024.06.29.601251) reports that RNAs with G-quadruplexes\n(rG4) bind both ADAR1 p150 and ZBP1 via their Zα domains, further undermining\nthe novelty of this manuscript.\nResponse:\nWe appreciate the reviewer's feedback, which helps us clarify the novel aspects\nof our work. We agree that the general ability of Zα domains to bind G4 structures is\nrecognized, as evidenced by the studies cited. We would like to emphasize that the\ncore novelty of our study lies in identifying a novel upstream signal for ZBP1\nactivation within the specific chromosomal context of the telomere and involving the\nkey non-coding RNA, TERRA. Previous studies, including the mentioned preprint,\nhave largely focused on in vitro identification or genome-wide binding of rG4s.\nIn contrast, our work is the first to demonstrate that endogenous telomeric RNA\n(TERRA), in its G-quadruplex form, can specifically recruit and activate ZBP1 in\nliving cells. The significance of this finding is that it defines a spatially restricted\ninnate immune signaling pathway triggered by a precisely localized 'addressing' G4\n(TERRA at telomeres). This stands in clear contrast to the role of ADAR1 p150 in\nbroader antiviral responses. Thus, we believe the conceptual advance here is the\ndiscovery of a mechanism for the precise initiation of cell death signaling, guided by a\nlocation-specific RNA G4, opening new avenues for understanding the crosstalk\nbetween telomere biology and innate immune signaling.\n2.Weak Experimental Support for Key Claims\n1）Fig. 2f: The interaction between TERRA G4 and ZBP1-S appears extremely weak.\nResponse:\nThank you for your valuable suggestions. In the revised manuscript, we have\nrepeated the key binding assay under optimized conditions, and the new data, now\npresented in the revised Fig. 2f, shows a tight interaction between TERRA G4 and\nZBP1-S. More importantly, we have performed additional, orthogonal experiments to\nvalidate the specificity of this interaction. The supershift assay using a validated\nanti-ZBP1 antibody demonstrates a definitive complex formation between TERRA\nG4 and ZBP1-S (Fig. 2k). We also performed a competitive binding assay using\nunlabeled TERRA G4, which confirms the specificity of the observed interaction (Fig.\n2g). And The point mutations (S106A, Q107A, R142A and R150A) block the direct\nand specific interaction between TERRA G4 and ZBP1-S (Fig. 2j). Consistently, a\npulldown assay employing biotinylated TERRA G4 RNA confirmed the interaction\nbetween TERRA G4 and ZBP1 in living cells (Extended Data Fig. 2e). The\nconvergence of evidence from these multiple, independent experimental approaches\nprovides strong and compelling support for the direct and specific interaction between\nTERRA G4 and ZBP1-S. We believe that these new data resolve any ambiguity and\nsolidly underpin our conclusion.\nThe renewed and added results were described on page 6 and 7 of the revised\nmanuscript (Fig. 2f, 2g, 2j and 2k; Extended Data Fig. 2e). The description\nhighlighted in yellow.\n2）Fig. 2i: The representative images show no clear difference in colocalization\nbetween G4 WT and G4-mut with ZBP1.\nResponse:\nThank you for your comments on the representative images in Fig. 2i (Fig. 2l in\nthe revised manuscript). We agree that the selected images may not, on their own,\nconvey a dramatic qualitative difference. This is a common challenge in imaging\nstudies, where the intensity and distribution of signals can vary from cell to cell, and a\nsingle snapshot may not fully capture the quantitative trend observed across the entire\ndataset. The mutant G4 (G4-mut) probe does exhibit a low level of\nbackground/non-specific localization, as the reviewer noted, which is expected in\ncellular imaging experiments. However, the key conclusion that the wild-type\nTERRA G4 (TERRA-G4 WT) probe exhibits significantly stronger colocalization\nwith ZBP1 is drawn not from a single representative image, but from the rigorous\nquantitative analysis of a certain number of cells (n =10). The statistical analysis (p <\n0.001) unambiguously demonstrates that the colocalization between TERRA-G4 WT\nand ZBP1 is significantly greater than that between TERRA-G4 Mut and ZBP1. We\nhave greater confidence in this objective, population-based measurement than in\nsubjective visual interpretation of individual micrographs. Therefore, the quantitation\nin Fig. 2l provides the definitive evidence for our claim. In the revised manuscript, we\nincluded additional representative images in the revised manuscript (Extended Data\nFig. 2f).\n3.The study relies entirely on synthetic nucleic acids, overexpression, and\ntransfection assays, which are highly artificial. There is no evidence that endogenous\nG4 structures adopt a Z-form conformation and activate ZBP1 under physiological\nconditions.\nResponse:\nThank you for your comments. In this report, we would like to clarify a key\naspect of our proposed mechanism. Our data suggest that ZBP1 is activated by direct\nrecognition of the G4 structure itself, not because the G4 adopts a Z-form\nconformation. The Zα domain is known to bind to both Z-form nucleic acids and\ncertain non-B DNA/RNA structures, including G4s. Our model shows that the\nTERRA G4 serves as a distinct, high-affinity ligand for ZBP1's Zα domain, triggering\nits activation. Therefore, the question is not about G4s converting to Z-DNA/RNA,\nbut about whether endogenous G4s naturally engage with ZBP1. In this context, the\nuse of synthetic G4s and overexpression systems was a necessary and reductionist\napproach to first rigorously establish this direct, specific interaction and its functional\nconsequences, free from the complexities of the endogenous cellular environment.\nThese controlled conditions allow us to verify causality.\nFurthermore, we also employed a complementary, physiologically relevant\napproach to probe the endogenous pathway. Specifically, we perturbed the\nendogenous TERRA G4-ZBP1 interaction by using G4 ligands PDS and TMPyP4.\nWe found that treatment with these G4 ligands can significantly suppress the\nactivation of ZBP1. In addition, we investigated whether RNA helicases such as\nDHX36 could influence ZBP1-mediated activation of innate immune signaling by\nunwinding endogenous TERRA G4s. As expected, DHX36 knockdown significantly\nincrease the expression of ISGs in cells co-overexpressing ZBP1-S and TERRA\n(Extended Data Fig. 9a,b), indicating further activation of ZBP1-mediated innate\nimmune activation. These results suggest that DHX36 acts as a negative regulator of\nthis pathway. We proposed a model wherein DHX36 normally functions to unwind or\nremodel TERRA G4s, thereby dismantling the activating ligand for ZBP1 and\nsuppressing signaling.\nThe added results were described on page 9 and 10 of the revised manuscript\n(Extended Data Fig. 9a,b). The description highlighted in yellow.\n4.Fig. 3d & 3f: The reported phenotypes are barely detectable, making it difficult to\nsupport the authors’ conclusions.\nResponse:\nThank you for your valuable suggestions. We sincerely apologize that the\nphenotypes were not easily detectable in our initial submission; this was an oversight\nin our graphical presentation. The quantitative measurements for these experiments\nwere indeed statistically significant. However, we recognize that the graphical\nrepresentation is crucial for the reader. In the revised manuscript, we have taken\nseveral steps to improve these figures, including: Changing the plot type to better\nhighlight the differences between groups; Adjusting the scale and labels to reduce\nvisual clutter and improve clarity. We are confident that the revised figures now\nprovide a transparent and compelling visual representation of our data (Fig. 3e and 3g\nin the revised manuscript), which robustly supports our conclusions. Thank you for\nsuggesting this important improvement.\n5. Fig. 3 (ISG induction): The fold changes in ISGs are minimal, suggesting that\nTERRA G4 has only a marginal effect on ZBP1 activation.\nResponse:\nThank you for your comments. We agree that the fold-changes in Fig. 3 are more\nmodest than those reported in the seminal Nassour et al. (Nature. 2023\nFeb;614(7949):767-773) study. We reasoned that this quantitative difference was\nunlikely to reflect an intrinsic weakness of the TERRA G4-ZBP1 pathway, but rather\na cell line-specific variation in the baseline state or responsiveness of the innate\nimmune system.\nTo directly address this point, we followed the reviewer's implicit suggestion to\ninvestigate the robustness of our findings. We have now repeated these key\nexperiments in several additional cell lines, such as Hela and A549 cells. As\nanticipated, we observed a much more pronounced induction of ISGs in certain cell\nbackgrounds, unequivocally demonstrating that the TERRA G4-ZBP1-MAVS axis is\ncapable of driving robust interferon responses. These new data are included in the\nrevised manuscript as Extended Data Fig. 5f,g. Therefore, we conclude that the\neffect we initially reported is genuine and not marginal. The variability across cell\nlines underscores the biological reality of this pathway and highlights that cellular\ncontext is a critical determinant of the signaling output.\nThe renewed and added results were described on page 8 of the revised\nmanuscript (Extended Data Fig. 5f,g). The description highlighted in yellow.\n6. While Figures 2C-D suggest novel binding sites through structural modeling, the\nauthors did not experimentally validate these predictions. Instead, they proceeded\nwith Zα binding site mutants, a logical leap that lacks sufficient mechanistic support.\nKey concerns include: the assumed (but unproven) similarity between TERRA G4\nand Z-form DNA. The failure to follow up on predicted critical residues (e.g., N141,\nY145) implicated by their own structural data.\nResponse:\nThank you for your insightful comments on insight. The reviewer is correct that\nour initial submission relied on structural modeling and then proceeded to canonical\nZα mutants without fully validating the novel binding interfaces predicted by our own\nmodel. In direct response to this comment, we have now performed the essential\nexperimental validation. As suggested, we generated the point mutants targeting the\nspecific residues (S106, Q107, R142, and R150 in ZBP1; corresponding to S8, Q9,\nR44, and R52 in the Zα domain) that our structural model predicted to be critical for\nTERRA G4 binding. These new data are now included in the revised manuscript as\nFig. 2j,k and Extended Data Fig. 2c,d. The results are clear and definitive: mutation\nof these predicted residues significantly disrupts the interaction between ZBP1-S and\nTERRA G4 in our in vitro binding assays. This experimental follow-up directly\nbridges the gap between our computational predictions and functional data. It\nprovides strong mechanistic support for our model, demonstrating that these specific\nresidues form a novel binding interface distinct from the canonical Z-form nucleic\nacid binding mode, and that this interface is essential for TERRA G4 recognition. We\nare grateful to the reviewer for guiding us to make this crucial addition, which has\nsubstantially strengthened the mechanistic foundation of our study.\nThe renewed results were described on page 6 and 7 of the revised manuscript\n(Fig. 2j,k and Extended Data Fig. 2c,d). The description highlighted in yellow.\n7. Several experiments lack essential controls. For example, Extended Data Fig. 2C-D\nomits a direct comparison between ZBP1-S WT and Mut, which is crucial for\ninterpreting functional differences.\nResponse:\nThank you for your helpful comments. We agree that a direct comparison\nbetween ZBP1-S WT and Mut is essential for a clear interpretation of the functional\ndata. As recommended, we have now renewed the experiments in Extended Data Fig.\n2c,d to include a side-by-side comparison of the ZBP1-S WT and Mut. The new data\nprovide a much clearer and more direct demonstration of the functional differences\nbetween the WT and Mutant ZBP1-S, unequivocally supporting our conclusions. Zα2\nmutant impaired the binding of ZBP1-S to TERRA G4. We believe these revised\nfigures now meet the necessary standard for interpretation, and thank the reviewer for\nthe suggestion, which has significantly strengthened our manuscript.\nThe renewed results were described on page 6 and 7 of the revised manuscript\n(Extended Data Fig. c,d). The description highlighted in yellow.\n8. The study reports that ZBP1-S RHIM2 is necessary for inflammation and cell death\n(Extended Data Fig. 4, 5E), contradicting prior work suggesting RHIM2 is largely\ndispensable. Whether this discrepancy reflects a context-dependent phenomenon or\nmethodological differences remains unclear.\nResponse:\nWe are extremely grateful to the reviewer for the comments regarding the role of\nthe RHIM2 domain. The reviewer identified the discrepancy with the established\nliterature, which prompted us to conduct a thorough investigation into our\nexperimental materials and data. This investigation revealed that the original RHIM2\nmutant construct was erroneously built, which led to the incorrect conclusion about its\nnecessity. We have since re-generated the correct RHIM2 mutant construct and\nrepeated the relevant experiments multiple times. The new, validated data consistently\nshow that the RHIM2 domain plays, at best, a minor role in our system, which is now\nfully aligned with prior reports indicating it is largely dispensable. We have therefore\nremoved the original, non-reproducible data from Extended Data Fig. 4c,d and 5e\nand replaced them with the corrected results in the revised manuscript. All related\nconclusions in the text have been updated accordingly. We sincerely apologize for\nthis error. We thank the reviewer again for their critical insight, which has\nsignificantly strengthened the study.\nThe renewed results were described on page 8 of the revised manuscript\n(Extended Data Fig. 4c,d and 5e). The description highlighted in yellow.\nReviewer #2:\nThe study by Qin et al. provides important mechanistic insight into how the short,\nhuman-specific isoform of ZBP1 (ZBP1-S) recognizes telomeric repeat-containing\nRNA (TERRA) and activates MAVS-dependent antiviral signaling. ZBP1 is known to\ncontain tandem Zα domains that bind left-handed Z-DNA and Z-RNA; however, it\nhas remained unclear how these domains could recognize single-stranded RNAs such\nas TERRA. Building on prior work by Nassour et al. (Nature, 2023), which\ndemonstrated that ZBP1-S senses TERRA and triggers interferon (IFN) signaling and\ncell death during telomere crisis, this study reveals that TERRA adopts G-quadruplex\n(G4) RNA structures that are directly bound by the Zα2 domain of ZBP1-S. This\ninteraction is shown to be both necessary and sufficient to drive downstream immune\nsignaling. Notably, this is the first demonstration of a Zα domain directly engaging an\nRNA G-quadruplex, thereby expanding the known ligand repertoire of Zα domains\nand identifying G4 RNA as a novel class of immunostimulatory structures. While\nprevious studies have shown that ZBP1 can be activated by viral infections and\nendogenous retroelements (EREs) through recognition of Z-form nucleic\nacids—including Z-RNAs produced during influenza, vaccinia virus infection, and\nretrotransposon expression—this work raises the intriguing possibility that structured\nviral RNAs forming G4 motifs may also serve as ligands for ZBP1-S. However, to\nmove beyond a conceptual extension of the previously defined TERRA–ZBP1 axis,\nthe study must more thoroughly define the molecular basis of this\ninteraction—particularly the structural diversity of G4 RNAs involved, whether\nmultimeric or stacked G4 assemblies are required to promote ZBP1 multimerization\nand filament formation, and how ZBP1 oligomerization is initiated, stabilized, and\nspatially organized on these RNA scaffolds. In addition, the specificity of ZBP1-S for\nG4 RNAs, as compared to other structured or unstructured RNA species, remains to\nbe determined. It also remains to be clarified whether ZBP1 assembles into\nfilament-like oligomers along RNA templates, similar to the cooperative filament\nformation observed for MDA5 during sensing of long dsRNA. Without addressing\nthese mechanistic questions, the study risks being interpreted as a refinement of\nearlier findings rather than a significant conceptual advance. Nevertheless, the\nidentification of RNA G-quadruplexes as functional ligands for ZBP1-S represents a\nnovel and biologically meaningful development in the field of RNA structure–driven\ninnate immunity, and clearly merits deeper investigation.\nResponse:\nWe are deeply grateful to the reviewer for their exceptionally thoughtful and\npositive assessment of our work, and for their insightful suggestions which have\nsignificantly helped us in strengthening the manuscript. We are particularly\nencouraged by their recognition that our study \"provides important mechanistic\ninsight\". We thank the reviewer for the time spent on reviewing our manuscript, and\nthe insightful comments. The inputs and suggestions from the reviewer have been\nimplemented in the manuscript. Please see our point-to-point response below.\n1. Validation of ZBP1–TERRA G4 binding\nThe interaction between ZBP1-S and TERRA G4 RNA lies at the core of the\nmanuscript’s mechanistic claims. While the EMSA results suggest a Zα2-dependent\ninteraction, several technical issues limit interpretation. The free RNA band is heavily\noverexposed, and shifted complexes are faint and poorly resolved—especially at\nlower ZBP1 concentrations. Furthermore, minimal reduction in free RNA suggests\nweak binding efficiency or suboptimal assay conditions. I recommend repeating the\nEMSA with optimized RNA:protein ratios and improved imaging for better contrast\nand resolution. A supershift assay using a validated anti-ZBP1 antibody (e.g., CST\n#60968) would help confirm complex identity. Inclusion of non-G4 RNA controls and\ncompetition EMSAs with unlabeled TERRA would also strengthen specificity claims.\nFinally, a pulldown assay using biotinylated G4 RNA with ZBP1 Western blot\ndetection would provide orthogonal confirmation.\nResponse:\nThank you for your critical and constructive suggestions regarding the EMSA\ndata. We agree that the initial conditions and presentation limited the clarity and\ninterpretation of the results. In direct response to the reviewer's recommendations, we\nhave comprehensively revised this part of our study:\n(1) We have repeated the EMSA under optimized conditions, using adjusted\nRNA:protein ratios and improved imaging parameters. The new gels, now presented\nin Fig. 2f and 2j, show well-resolved bands with significantly improved contrast,\nclearly demonstrating the concentration-dependent formation of the shifted\ncomplexes.\n(2) To unambiguously confirm the identity of the protein in the shifted complex,\nwe have performed a supershift assay using the validated anti-ZBP1 antibody. The\nsuccessful supershift, included in Fig. 2k, provides definitive evidence that ZBP1-S is\nspecifically bound to the TERRA G4 RNA.\n(3) To rigorously establish the specificity of the interaction, we have now\nincluded crucial controls as suggested: A non-G4 RNA control, which shows no\nsignificant binding to ZBP1-S (Fig. 2f). A competition EMSA with unlabeled\nTERRA G4 RNA. The TERRA G4 effectively competes away the binding,\nconfirming the sequence and structure specificity of the interaction (Fig. 2g).\n(4) A pulldown assay employing biotinylated TERRA G4 RNA confirmed the\ninteraction between TERRA G4 and ZBP1 in living cells (Extended Data Fig. 2e).\nWe believe that this suite of new, orthogonal data—the optimized EMSA,\nsupershift, and specificity controls—collectively provides robust and conclusive\nevidence for the direct and specific interaction between ZBP1-S and the TERRA G4,\nfully addressing the reviewer's concerns. We are grateful for these insightful\ncomments, which have substantially strengthened the mechanistic foundation of our\nmanuscript.\nThe renewed results were described on page 6 and 7 of the revised manuscript\n(Fig. 2f, 2g, 2j and 2k; Extended Data Fig. 2e). The description highlighted in\nyellow.\n2.Evidence for ZBP1 oligomerization\nThe claim that ZBP1-S forms filament-like assemblies is currently based on\nimmunofluorescence, but the resolution is insufficient to distinguish true filaments\nfrom puncta or aggregates. Some figures even suggest primarily punctate localization\nwith TERRA. A plausible model is that ZBP1 first binds TERRA G4 RNA to form\npuncta, and then oligomerizes into filaments along the RNA scaffold, as seen with\nMDA5 and other sensors. This hypothesis should be explicitly tested and depicted.\nHigher-resolution microscopy (e.g., SIM, Airyscan, STED) would help clarify\nmorphology. Biochemical validation is also necessary: native or blue native PAGE,\ncrosslinking, or SEC-MALS could reveal oligomerization states. Reconstitution with\nsynthetic G4s could test whether multimeric G4 architecture promotes ZBP1 filament\nformation. If ZBP1 does form extended assemblies, cryo-EM or negative-stain EM\nwould provide valuable structural insight.\nResponse:\nThank you for the insightful comments. We agree that higher-resolution imaging\nand structural studies would provide invaluable insight into the precise architecture of\nthese complexes. In our study, we focused on establishing the upstream event-the\ndirect recognition of TERRA G4 by ZBP1's Zα domain as the initial trigger.\nRegarding the subsequent oligomerization, we are guided by the elegant model\nestablished in the seminal work by Nassour et al. (Nature. 2023\nFeb;614(7949):767-773). Their data indicate that ZBP1's oligomerization is distinct\nfrom the cooperative filament formation seen with MDA5 along a long dsRNA\nscaffold. Instead, the current model posits that TERRA binding induces a\nconformational change in ZBP1, which then facilitates its translocation and\nsubsequent filamentous assembly on the mitochondrial membrane in conjunction with\nMAVS, rather than forming extensive filaments along the RNA itself. We fully\nacknowledge that our current immunofluorescence data cannot definitively resolve\nthe ultrastructure of these assemblies. The reviewer's suggestions regarding\nhigh-resolution microscopy are highly valuable and represent logical next steps\ntoward determining the precise structural interplay between ZBP1 and MAVS. In the\nfuture, we will conduct an in-depth investigation into this issue.\n3.Broader characterization of Zα domain ligand specificity\nGiven that ADAR1’s Zα domain can bind G4 DNA, it would be highly informative to\ntest whether ZBP1’s Zα domains can also bind structured DNA—including G4\nDNA—or other nucleic acid types. Comparative binding across ssRNA, dsRNA,\nssDNA, dsDNA, RNA:DNA hybrids, and G4 DNA would clarify whether ZBP1 is\nemerging as a general nucleic acid sensor. This would significantly expand the\nfunctional understanding of ZBP1-S and help position it within the broader landscape\nof innate immune pattern recognition receptors.\nResponse:\nThank you for your excellent and forward-looking suggestions. We agree that a\nbroader characterization of ZBP1's Zα domain specificity is crucial for understanding\nits role in innate immunity. Following the advice, we compared the affinity of the\nZBP1 Zα domain for a wide range of nucleic acid substrates, including ssRNA,\ndsRNA, ssDNA, dsDNA, and two G4 DNAs (including the myc and KRAS promoter\nG4 DNAs). We found that the ZBP1 Zα domain binds robustly to G4 DNA structures\n(such as mycG4 and KRAS G4), consistent with its binding to TERRA G4 RNA.\nHowever, it showed no significant binding to the other tested substrates, including\nssRNA, dsRNA, ssDNA, and dsDNA (Extended Data Fig. 11a-d). This new data\ndemonstrates that ZBP1 is not a general nucleic acid sensor but is instead highly\nspecific for Z-DNA, Z-RNA and G4 DNA/RNA. We have updated the manuscript\naccordingly.\nThe renewed results were described on page 10 of the revised manuscript\n(Extended Data Fig. 11a-d). The description highlighted in yellow.\n4.Mechanistic comparison with RIG-I and MDA5\nThe authors should more explicitly address whether ZBP1 follows a mechanism more\nanalogous to RIG-I (short dsRNA, polyubiquitin-assisted tetramerization) or MDA5\n(long dsRNA-guided filament formation). This comparison would help\ncontextualize the findings and clarify whether ZBP1 represents a distinct G4-based\nsensing pathway.\nResponse:\nThank you for this insightful question, which allows us to clarify a key\ninnovative aspect of our study. Based on our data and the foundational model\nestablished by Nassour et al. (Nature. 2023 Feb;614(7949):767-773), ZBP1 operates\nvia a mechanism distinct from both RIG-I and MDA5. It is not activated by short\ndsRNA like RIG-I, nor does its oligomerization seem to be primarily driven by\ncooperative filament formation along a long, repetitive dsRNA scaffold like MDA5.\nInstead, the current model suggests that ZBP1 is activated by a specific RNA\nstructure—TERRA G4. Binding to this ligand is proposed to induce a conformational\nchange in ZBP1, which licenses its subsequent oligomerization and signaling,\npotentially in a manner that is less dependent on the length of the RNA and more on\nthe presence of discrete, high-affinity G4 structural elements. Therefore, we posit that\nZBP1 represents a distinct class of innate immune sensor that is defined by its\nspecificity for nucleic acid secondary structures (Z-form and G4), setting it apart from\nthe dsRNA-length sensors RIG-I and MDA5. In the revised manuscript, we have now\nadded the discussion to include a more explicit comparison with RIG-I and MDA5,\nframing ZBP1 as a member of a growing family of pattern recognition receptors that\nare tuned to specific nucleic acid conformations rather than just their sequence or\nlength.\nThe discussion was added on page 11 of the revised manuscript. The discussion\nhighlighted in yellow.\n5.Role of multimeric G4 structures\nRNA G4s exhibit diverse topologies, including monomeric, stacked, and\nintermolecular forms. The manuscript would benefit from deeper biophysical\ncharacterization of the TERRA G4s used—via CD spectroscopy, native PAGE, or\nThT/NMM fluorescence. Comparative assays with synthetic G4s of defined\narchitecture (e.g., dimers, trimers, loop-length variants) could test whether ZBP1\ndistinguishes between them. This would clarify whether G4 multimerization state\ninfluences ZBP1 activation or oligomerization.\nResponse:\nThank you for the insightful comments regarding the potential role of G4\nmultimerization, a fascinating aspect of G4 biology. We agree that a deeper\nbiophysical characterization is valuable. In our study, we have performed biophysical\nanalysis of TERRA G4 dimers using Circular Dichroism (CD) spectroscopy and\nnative PAGE, confirming the formation of a stable, parallel-stranded G-quadruplex\nunder our experimental conditions (Extended Data Fig. 1f-h). The reviewer's\nsuggestion to test trimers or higher-order structures is highly pertinent. However, the\nin vitro reconstitution of defined, homogeneous, and stable RNA G4 trimers remains a\nsignificant technical challenge in the field, as these sequences are prone to\naggregation and heterogeneity. This currently limits the systematic study of such\narchitectures. Therefore, while our study provides a foundational understanding using\nmonomeric and dimeric TERRA G4s, we fully acknowledge that the role of\nhigher-order multimerization is an open and compelling question. We have framed the\npursuit of defined multimeric G4 structures as a critical goal for our future research.\nWe are grateful for this suggestion, which helps chart the course for ongoing\ninvestigations.\n6. Split-GFP assay: The split-GFP results for ZBP1-S oligomerization are ambiguous.\nThe GFP signal appears diffusely nuclear and cytoplasmic, rather than as discrete\npuncta or filaments. This undermines the conclusion that ZBP1 oligomerizes in a\ntemplate-dependent manner. The authors should clarify this interpretation, possibly\nusing higher-resolution imaging or quantification.\nResponse:\nThank you for this thoughtful comment regarding the interpretation of our\nsplit-GFP data. We agree that the GFP signal appears diffuse and does not resolve\ninto distinct puncta or filaments under conventional microscopy. However, the\nfundamental principle of the split-GFP system is that fluorescence complementation\noccurs only when the two fragments are brought into close proximity by the\ninteraction and oligomerization of the proteins to which they are fused. Therefore, the\npresence of a robust fluorescent signal, even if diffuse, provides direct and qualitative\nevidence that ZBP1-S molecules are interacting and oligomerizing in live cells. This\nis a well-established and widely accepted interpretation of such assays. The diffuse\nnature of the signal, as opposed to the formation of sharp puncta, is a common\nobservation in split-GFP and other protein-fragment complementation assays. This\ncan be attributed to several factors, including the stability of the reconstituted GFP\nmolecule, which can diffuse within the cell after complementation, and the potential\nthat the GFP tags themselves, due to their size or charge, might interfere with the\nhigher-order packing or subcellular localization of the oligomerizing complex. Indeed,\nsimilar diffuse signals for validated ZBP1 oligomerization have been reported in the\nliterature (bioRxiv. 2024 Jun 30:2024.06.29.601251). While we agree that\nhigher-resolution imaging of these complexes represents a compelling objective, the\ncurrent split-GFP data robustly support our central conclusion that ZBP1-S undergoes\nligand-induced oligomerization. We thank the reviewer for this suggestion. Although\nhigher-resolution imaging is not currently feasible in our experimental system, the\nsplit-GFP signal remains a valid and reliable indicator of oligomerization.\n7. ZBP1 knockout validation: The use of ZBP1-KO MCF-7 cells is a key\nexperimental component, but no data are shown to confirm knockout efficiency.\nAt minimum, a Western blot demonstrating loss of endogenous ZBP1 is required.\nResponse:\nThank you for pointing out this essential control. In response, we have now\nincluded a Western blot analysis in the revised manuscript as Extended Data Fig. 3a\nthat clearly demonstrates the loss of endogenous ZBP1 protein in our ZBP1-KO\nMCF-7 cell line, thereby validating its use in our functional experiments.\nThe added results were described on page 8 of the revised manuscript (Extended\nData Fig. 3a). The description highlighted in yellow.\n8. Figure legends and replicates: Several figure legends lack clarity, and the number\nof biological replicates is not consistently reported. Please revise legends to include\nkey experimental details, number of replicates, and statistical analyses.\nResponse:\nThank you for your suggestion to improve the clarity and reproducibility of our\ndata. In response, we have meticulously updated all figure legends to consistently\nreport the number of biological replicates and key methodological information. We\nbelieve these revisions have significantly enhanced the transparency of our study.\n9. This study addresses a biologically compelling and timely question by investigating\nhow the human-specific ZBP1-S isoform senses structured RNA to activate innate\nimmunity. While ZBP1 has been extensively studied in the context of necroptosis in\nmice, its activation mechanisms in humans remain underexplored. The identification\nof TERRA G4 RNA as a functional ligand for ZBP1-S via a Zα domain is a notable\nconceptual advance. However, to fully realize the impact of this discovery, additional\nclarification is needed on (1) the structural nature and specificity of the\nZBP1–TERRA G4 interaction, and (2) biochemical and structural evidence for ZBP1\noligomerization. The authors are well-positioned to address these points, and doing so\nwould significantly strengthen the manuscript and offer foundational insights into a\nnovel human innate immune mechanism.\nResponse:\nWe are profoundly grateful to the reviewer for their exceptionally generous and\ninsightful assessment of our work. Their recognition that our study addresses a\n\"biologically compelling and timely question\" and represents a \"notable conceptual\nadvance\" is deeply encouraging. We also wish to express our sincere thanks for their\nconstructive guidance in identifying the key areas for strengthening the manuscript. In\ndirect response to their points, we have undertaken significant revisions to provide\ngreater clarification on both the structural specificity of the ZBP1–TERRA G4\ninteraction and the biochemical evidence for ZBP1 oligomerization, which we believe\nhave substantially strengthened the foundational insights of our work. The reviewer's\npositive engagement and expert critique have been invaluable in shaping the final\nversion of this manuscript, and many thanks for your insightful comments and\nsuggestions for improving our work quality and potential impact.\nReviewer #3:\nIn the submitted manuscript, Qin et al. reported that ZBP1-S interacts with\nTERRA G-quadruplexes (G4s) through its Z-DNA-binding domains (Zα). TERRA\nG4 structures are critical for ZBP1-S-mediated, MAVS-dependent interferon\nresponses and inflammatory cell death. G4-specific ligands (PDS and TMPyP4)\ncompetitively inhibit ZBP1-S binding to TERRA G4s, thereby suppressing\nZBP1-S-mediated activation of interferon signaling. In contrast, TERRA G4\noligonucleotides can induce ZBP1-S-mediated inflammatory cell death. These\nfindings elucidate the precise mechanism by which ZBP1-S recognizes TERRA,\nhighlighting the essential role of G4 structures in ZBP1-mediated activation of innate\nimmune signaling and inflammatory cell death.\nAfter reading this work, my feeling is taht the work is novel and important to\nunderstanding G4 biology and ZBP1 mediated innate immune signaling and\ninflammatory cell death. The authors have employed appropriate experimental\ncontrols and applied suitable statistical analyses to support their key conclusions, thus\nI am pleased to recommend the acceptance of this manuscript for publication in\nNature Communications. The major and minor comments of this work were outlined\nbelow, which I believe these would further enhance the clarity and impact of the\nmanuscript.\nResponse:\nWe thank the reviewer for the time spent on reviewing our manuscript, and the\ninsightful comments. The inputs and suggestions from the reviewer have been\nimplemented in the manuscript. Please see our point-to-point response below.\n1.The author demonstrated that ZBP1-S recognizes TERRA G4 structures, thereby\nactivating ZBP1-S- and MAVS-dependent interferon responses and inflammatory cell\ndeath. It is known that the recognition of endogenous Z-form nucleic acids by ZBP1\ninitiates RIPK3-dependent necroptosis and inflammation. This raises the question:\ndoes the sensing of endogenous G4 structures by ZBP1 also induce RIPK3-dependent\nnecroptosis and inflammation?\nResponse:\nThank you for the insightful question regarding the downstream consequences of\nG4 sensing by ZBP1. The reviewer is correct that the canonical pathway for ZBP1\nactivation by Z-form nucleic acids is through RIPK3-dependent necroptosis and\ninflammation. However, our data clearly demonstrate that the sensing of TERRA G4\nstructures by ZBP1 engages a distinct signaling axis. In our models, we find that\nZBP1-dependent inflammatory and cell death responses triggered by TERRA G4 are\nindependent of RIPK3. Instead, they rely on the signaling adaptor MAVS to drive\ninterferon responses. This observation is not without precedent and aligns with\nemerging concepts of ZBP1 signaling plasticity. Importantly, our findings are strongly\nsupported by the seminal work by Nassour et al. (Nature. 2023\nFeb;614(7949):767-773), which elegantly demonstrated that ZBP1 activation by\nterminal telomeric repeats signals via MAVS and not via RIPK3 to eliminate cells in\ncrisis. Our study builds upon this paradigm by identifying the specific ligand at the\ntelomere—the TERRA G4—that initiates this non-canonical, RIPK3-independent\npathway. Therefore, we propose that the nature of the nucleic acid ligand (Z-form vs.\nG4) and its cellular context (e.g., telomeres) can dictate the choice of downstream\nsignaling partners, unveiling a previously underappreciated layer of specificity in\ninnate immune sensing.\n2.Only two proteins containing Zα domains have been identified in mammals:\nADAR1 and ZBP1. A pertinent question arises: does ADAR1 recognize TERRA\nG-quadruplex (G4) structures? If so, what is the functional relationship between ZBP1,\nADAR1, and TERRA G4?\nResponse:\nThank you for raising this pertinent and insightful question regarding the\npotential role of ADAR1 in TERRA G4 recognition and its functional interplay with\nZBP1. We agree that this is a critical aspect of the biology of Zα domain-containing\nproteins, and we have indeed dedicated a portion of our Discussion to this very topic.\nBased on prior literature and our own findings, we propose a model where ADAR1 is\nlikely involved in regulating the TERRA G4-ZBP1 signaling axis, but primarily as a\nmodulator rather than the primary sensor in this specific context. As the reviewer\nalludes to, several studies (Nature. 2022 Jun;606(7914):594-602; Nature. 2022\nJul;607(7920):769-775; Nature. 2022 Jul;607(7920):784-789; Nature. 2022\nJul;607(7920):776-783) have established that ADAR1 functions as a potent negative\nregulator of ZBP1 activation, often by editing or competing for shared nucleic acid\nligands to prevent inadvertent innate immune activation. In the specific case of\nTERRA G4 signaling, it is plausible that ADAR1 p150 could bind TERRA G4s.\nHowever, our functional data indicate that under the conditions of cellular stress\nstudied here, ZBP1 emerges as the dominant sensor that triggers the downstream\nMAVS-dependent interferon and cell death response. We hypothesize that ADAR1\nmay serve a homeostatic role, perhaps binding to TERRA G4s under normal\nconditions to suppress signaling. Upon specific insults (such as the ones induced in\nour study), this suppression might be overcome, leading to robust ZBP1 activation.\nTherefore, while we fully acknowledge the likely involvement of ADAR1 in this\nregulatory network, the central and novel conceptual advance of our manuscript is the\nidentification of ZBP1 as the critical effector sensor that translates the TERRA G4\nsignal into a functional cellular outcome. Delineating the precise competitive or\nregulatory relationship between ADAR1 and ZBP1 at telomeric G4s presents an\nexciting and important direction for our future research, and we thank the reviewer for\nhighlighting its significance.\n3.In Figure 4 and Extended Data Figure 8, the author demonstrated that PDS and\nTMPyP4 inhibit ZBP1-mediated activation of innate immune signaling by blocking\nthe interaction between Zα domains and TERRA G-quadruplex (G4) structures. How\ndo G4 ligands interfere with the interaction between Zα domains and TERRA G4?\nDo these ligands compete with TERRA G4 for binding to Zα domains?\nResponse:\nThank you for the excellent question regarding the mechanistic basis of how G4\nligands interfere with the Zα-TERRA G4 interaction. As the reviewer correctly infers,\nthe prevailing model for G4-binding ligands such as PDS and TMPyP4 is that they\nfunction by intercalating into or stacking onto the G-quadruplex structure, thereby\nstabilizing it. In the context of our study, we propose that this ligand-induced\nstabilization or structural alteration of the TERRA G4 creates a steric hindrance or\ninduces a conformational change that occludes the binding site for the Zα domain of\nZBP1. Crucially, our experimental data support a model of competitive inhibition. As\nshown in Fig. 4b and Extended Data Fig. 8a, b, the presence of PDS or TMPyP4\nsignificantly disrupts the binding between recombinant ZBP1 and the TERRA G4\nRNA in our in vitro pulldown assays. This direct biochemical evidence strongly\nsuggests that the G4 ligands and the ZBP1 Zα domain compete for access to the same\nor overlapping binding surfaces on the TERRA G4 structure. The ligands, by binding\nand stabilizing the G4, effectively prevent the subsequent recruitment of ZBP1.\nTherefore, we conclude that these G4 ligands do not directly bind to the Zα domain of\nZBP1 to antagonize the interaction. Instead, they act on the nucleic acid ligand itself,\ncompeting with ZBP1 for binding to the TERRA G4 and thereby inhibiting the\ndownstream innate immune signaling cascade.\n4.In Fig 2i, I believe it should be “FAM-G4-2” rather than “FAM-G4-4”?\nResponse:\nThank you for your careful and valuable suggestions. We apologize for the\nmistake. As recommended, we had corrected these mistakes in the revised manuscript.\n5.The author demonstrated that PDS and TMPyP4 block the interaction between Zα\ndomains and TERRA G4 structures. Can all G4 ligands exert such an effect? The\nauthor is encouraged to validate the effect using several additional G4 ligands.\nResponse:\nThank you for your constructive suggestion. We agree that investigating the\ngenerality of this effect across different G4 ligand classes is crucial for understanding\nthe specificity of the interaction. Following the reviewer's advice, we have now\nperformed additional experiments using several other structurally distinct G4 ligands,\nincluding PhpC, Phen-DC3 and BMVC. We are pleased to report that these new data,\nnow included in the revised manuscript as Extended Data Fig. 8d-g, provide a key\ninsight: not all G4 ligands are capable of disrupting the ZBP1-TERRA G4 interaction.\nSpecifically, we found that while ligands like PDS and TMPyP4 effectively inhibit\nthe binding, Phen-DC3 showed only a partial effect, and PhpC had no significant\ninhibitory effect under our experimental conditions. This important finding indicates\nthat the ability to interfere with ZBP1 recognition is not a universal property of G4\nligands but is highly specific. We hypothesize that this specificity may depend on the\nligand's binding mode, its preferred G4 topology, or the exact binding site it occupies\non the TERRA G4, which may or may not overlap with the Zα domain binding\ninterface. Therefore, the reviewer's suggestion has directly led us to a more nuanced\nconclusion: the ZBP1-TERRA G4 interaction can be competitively inhibited by a\nspecific subset of G4-stabilizing ligands, highlighting the structural specificity of this\nnucleic acid-protein interface. We are grateful for this input, which has significantly\nstrengthened our study.\nThe added results were described on page 9 of the revised manuscript (Extended\nData Fig. 8d-g). The description highlighted in yellow.\n6.Do RNA helicases, such as DHX36, influence ZBP1-mediated activation of innate\nimmune signaling through the unwinding of TERRA G4 structures?\nResponse:\nThank you for raising this insightful question regarding the potential role of\nRNA helicases. Following this suggestion, we investigated the role of the major\nG4-resolving helicase, DHX36. Strikingly, we found that knockdown of DHX36\nsignificantly potentiated the activation of the ZBP1-mediated innate immune\nsignaling pathway, which is now included in the revised manuscript as Extended\nData Fig. 9a,b. This result suggests that DHX36 acts as a negative regulator of this\npathway. We propose a model wherein DHX36 normally functions to unwind or\nremodel TERRA G4 structures, thereby dismantling the activating ligand for ZBP1\nand suppressing signaling. When DHX36 is depleted, these TERRA G4s become\nmore stable or persistent, leading to enhanced ZBP1 activation. This provides crucial\nfunctional evidence that the G4 structure itself is the critical ligand, and its integrity is\ndynamically regulated by cellular helicases. We have incorporated this new data and\nthe proposed model into the revised manuscript. We are grateful to the reviewer for\nthis suggestion, which has added a significant new layer of physiological regulation to\nour study.\nThe added results were described on page 9 and 10 of the revised manuscript\n(Extended Data Fig. 9a,b). The description highlighted in yellow.\n7.The quality of the Western blot results presented in Extended Data Figure 3d\nrequires improvement.\nResponse:\nThank you for your feedback on the quality of the Western blot. In response, we\nhave repeated the experiment under optimized conditions and have now included\nupdated, higher-resolution images for Extended Data Figure 3d (Extended Data\nFigure 3f in the revised manuscript). We believe the new data are of significantly\nimproved quality."}, {"round": 2, "reviewer_comments": {"reviewer_1": "While the authors have conducted additional experiments to improve the manuscript, the study in its current form still does\nnot adequately address several core concerns essential for validating its central claims and for publication. Major issues\npersist regarding the interpretation of data in the context of existing literature, the evidence for endogenous mechanisms,\nand the technical rigor of key assays.\nMajor Points:\n1.Apparent Conflict with Canonical ZBP1 Binding Mechanism.\nIn Fig.2, the authors claim that four residues (S106A, Q107A, R142A, R150A) in Zα2 define a novel binding interface for\nTERRA, distinct from the canonical Z-nucleic acid binding mode. However, this appears to conflict with the established\nmodel from Nassour et al. (Nature, 2023), which demonstrated that the canonical Zα2 domain mutation (N141A/Y145A)\nabrogates binding of TERRA-derived RNA and the subsequent interferon response. The authors must directly address this\ndiscrepancy. Is the proposed novel interface independent, cooperative, or contingent on the canonical binding mode?\nClarification on how these two binding models integrate is crucial.\n2.Evidence for the role of endogenous TERRA G4 is still lacking.\nA central claim of the manuscript is that endogenous TERRA, in its G4 form, recruits and activates ZBP1. However, all\nexperimental data provided rely on transfected, artificial G4 oligonucleotides. No evidence is presented to demonstrate that\nendogenous TERRA actually forms G4 structures in the cells under study, or that these putative endogenous G4s are\nresponsible for ZBP1 activation.\n3.Discrepancies between biochemical and cellular assays weaken the specificity claim.\nA significant inconsistency exists between the in vitro and cellular data. While the EMSA assays in Fig. 2 show a clear\nbinding defect for the FMA-G4-2-Mut, the representative cellular images in Fig. 2L show obvious colocalization of this same\nmutant with ZBP1-S, nearly indistinguishable from the wild-type probe. This discrepancy must be resolved, as it challenges\nthe specificity of the observed cellular interaction. Furthermore, the methodology for quantifying \"foci\" remains unclear and\nrequires a precise definition and a detailed description of the image analysis pipeline.\n4.Inadequate evidence for ZBP1-S oligomerization.\nThe evidence presented for ZBP1-S oligomerization is not convincing:\nFig. 3e, 3g (Split-GFP): The GFP signal is diffuse and does not form the discrete puncta or filaments expected for a template-\ndriven oligomeric assembly. This pattern does not robustly support the conclusion of specific oligomerization.\nQuantification and definition:\nThe criteria for identifying an \"oligomer\" in the immunofluorescence images (Fig. 3J) are not defined. The quantification\nmethod must be transparently described.\nNeed for complementary evidence:\nThe claims of oligomerization require validation through more definitive biochemical and biophysical methods. Suggestions\ninclude Native PAGE, crosslinking, or SEC-MALS to determine oligomeric states. Additionally, higher-resolution microscopy\n(e.g., SIM, Airyscan) would be necessary to properly visualize putative oligomeric structures.", "reviewer_2": "The revised manuscript by Qin et al. presents a substantially strengthened and well-executed study demonstrating that\nhuman ZBP1 recognizes telomeric TERRA RNA through RNA G-quadruplex (G4) structures to activate MAVS-dependent\ninnate immune signaling. The authors have addressed all major concerns from the initial review, and the new data provide\nrobust mechanistic support for their conclusions. The additional experiments, particularly the optimized EMSA and supershift\nassays confirming direct and specific interaction between ZBP1-S and TERRA G4 RNA, the inclusion of appropriate\ncompetition and non-G4 RNA controls, the validation of ZBP1 knockout cells, and the expanded ligand-binding analysis\ntesting the ability of Zα domains to interact with multiple nucleic acid types (ssRNA, dsRNA,...), collectively enhance the\nrigor of the study. The revised data provide convincing biochemical and functional evidence that TERRA G4 structures are\nspecifically recognized by the Zα domain of ZBP1 and that this interaction triggers MAVS-dependent innate immune\nsignaling and cell death. This finding is important because it expands the known spectrum of nucleic acid conformations\nsensed by ZBP1 beyond canonical Z-form structures, establishing G4 RNA as a new class of innate immune ligand and\nrevealing a direct molecular link between telomeric RNA architecture and antiviral and inflammatory signaling.\nMajor concern: The authors clarify that fluorescence complementation in the split GFP (BiFC) system reflects the proximity of\ntagged ZBP1 molecules and cite previous studies using this approach to infer oligomerization. While this rationale is\nunderstood, the method intrinsically cannot distinguish between direct ZBP1–ZBP1 interactions and the co-localization of\nmultiple ZBP1 molecules on the same multimeric or stacked G4 RNA scaffold. Although the BiFC data are consistent with\nligand-induced proximity of ZBP1 molecules, they do not by themselves demonstrate bona fide oligomerization. To\nstrengthen this conclusion, the authors could examine ZBP1 under RNA-free conditions using non-reducing, non-denaturing\nnative PAGE, which preserves weak intermolecular contacts and can reveal distinct higher molecular weight assemblies\nrelative to the monomer. Native PAGE has been successfully used to demonstrate oligomerization of other nucleic acid\nsensors and adaptors, including MAVS, and could similarly determine whether ZBP1 forms stable higher order assemblies.\nThis analysis would fill a key gap in the current study by providing direct biochemical evidence for ZBP1 self-association.\nMinor concern: The cell viability data currently presented in Extended Data Fig. 3 are essential for interpreting the functional\nconsequences of ZBP1 activation and should be quantified and incorporated into the main Figure 3. In addition, the image\nquality in Figure 3f is poor, and the visualization of filament-like ZBP1 structures is not convincing. The authors should\nimprove imaging resolution and include quantitative analysis of the proportion of cells exhibiting filamentous ZBP1\nstructures in Figure 3 to substantiate this observation.", "reviewer_3": "After reading the revised manuscript and the responses to all reviewers, I recommend the publication of the work in Nat. Commun."}, "author_response": "Next is the summarization of our point-by-point response to the comments raised by\nthe reviewers.\nReviewer #1:\nWhile the authors have conducted additional experiments to improve the\nmanuscript, the study in its current form still does not adequately address several core\nconcerns essential for validating its central claims and for publication. Major issues\npersist regarding the interpretation of data in the context of existing literature, the\nevidence for endogenous mechanisms, and the technical rigor of key assays.\nResponse:\nWe thank the reviewer for the time spent on reviewing our manuscript, and the\ninsightful comments. The inputs and suggestions from the reviewer have been\nimplemented in the manuscript. Please see our point-to-point response below.\n1.Apparent Conflict with Canonical ZBP1 Binding Mechanism.\nIn Fig.2, the authors claim that four residues (S106A, Q107A, R142A, R150A) in Zα2\ndefine a novel binding interface for TERRA, distinct from the canonical Z-nucleic\nacid binding mode. However, this appears to conflict with the established model from\nNassour et al. (Nature, 2023), which demonstrated that the canonical Zα2 domain\nmutation (N141A/Y145A) abrogates binding of TERRA-derived RNA and the\nsubsequent interferon response. The authors must directly address this discrepancy. Is\nthe proposed novel interface independent, cooperative, or contingent on the canonical\nbinding mode? Clarification on how these two binding models integrate is crucial.\nResponse:\nWe sincerely thank the reviewer for raising this critical and insightful point\nregarding the integration of our findings with the established canonical binding model.\nWe agree that reconciling these observations is paramount for a complete mechanistic\nunderstanding. Our data, consistent with the foundational work of Nassour et al.,\nconfirm that mutation of the canonical Zα2 residues (N141A/Y145A) also disrupts the\nTERRA G4-ZBP1 interaction in our assays. Therefore, our proposed model does not\nseek to replace the canonical framework but to expand upon it. The most parsimonious\ninterpretation is that the integrity of the entire Zα domain fold is essential for high-\naffinity G4 recognition. Mutations at key positions—whether the canonical\n(N141/Y145) or the ones we identified (S106/Q107/R142/R150)—likely induce local\nor global conformational changes that destabilize the binding interface. We speculate\nthat TERRA G4 engagement utilizes an extended or adapted binding surface on the Zα\ndomain, where both sets of residues contribute cooperatively to form a composite\ninterface tailored to the unique topology of the G4, distinct from a Z-form helix. Thus,\nthe binding modes are not independent but are integrated within a common structural\nscaffold. We have incorporated this discussion into the revised manuscript.\nFurthermore, we think that definitive mechanistic clarity will come from high-\nresolution structural studies. Determining the co-crystal structure of the Zα domain in\ncomplex with the telomeric repeat-containing RNA G-quadruplex, through\ncomparative analysis of wild-type and key mutant variants, represents a significant and\npromising direction for future research to visualize this interaction at atomic level.\nThe added discussion was described on page 12 of the revised manuscript. The\ndiscussion highlighted in yellow.\n2.Evidence for the role of endogenous TERRA G4 is still lacking.\nA central claim of the manuscript is that endogenous TERRA, in its G4 form, recruits\nand activates ZBP1. However, all experimental data provided rely on transfected,\nartificial G4 oligonucleotides. No evidence is presented to demonstrate that endogenous\nTERRA actually forms G4 structures in the cells under study, or that these putative\nendogenous G4s are responsible for ZBP1 activation.\nResponse:\nWe sincerely thank the reviewer for raising this crucial point regarding evidence\nfor endogenous TERRA G4s. We agree that demonstrating the existence and functional\nrelevance of these endogenous structures is paramount. In direct response to this\ncomment, we have performed new experiments to address this gap. Utilizing RNA\nimmunoprecipitation (RIP) with G4-specific antibody BG4, we now provide evidence\nthat a fraction of endogenous TERRA RNA indeed folds into G4 structures in our\ncellular models (Extended Data Fig. 3a). This finding directly supports the\nphysiological relevance of TERRA G4s.\nFurthermore, we acknowledge the limitations of using in vitro-transfected\nartificial G4 oligonucleotides or TERRA for research. We fully acknowledge, as the\nreviewer perceptively notes, a remaining technical challenge: the lack of tools to\nspecifically manipulate only the G4 conformation of endogenous TERRA without\naffecting its overall levels or sequence. This represents a current limitation not only for\nour study but for the broader field of endogenous G4 biology. We have explicitly\ndiscussed this limitation in the revised manuscript, emphasizing that while we can\nobserve these structures and perturb G4s globally with ligands, a definitive causal link\nspecifically from endogenous TERRA G4s to ZBP1 activation awaits the future\ndevelopment of precise, structure-specific manipulation strategies. We highlight this as\nan important direction for future research. We are grateful for the reviewer's suggestion,\nwhich has prompted us to strengthen our evidence for endogenous TERRA G4\nformation and to provide a more balanced and forward-looking perspective on the\nmechanistic conclusions of our work.\nThe added results were described on page 7 of the revised manuscript (Extended\nData Fig. 3a). The added discussion was described on page 13 of the revised\nmanuscript. The discussion highlighted in yellow.\n3.Discrepancies between biochemical and cellular assays weaken the specificity\nclaim.\nA significant inconsistency exists between the in vitro and cellular data. While the\nEMSA assays in Fig. 2 show a clear binding defect for the FMA-G4-2-Mut, the\nrepresentative cellular images in Fig. 2L show obvious colocalization of this same\nmutant with ZBP1-S, nearly indistinguishable from the wild-type probe. This\ndiscrepancy must be resolved, as it challenges the specificity of the observed cellular\ninteraction. Furthermore, the methodology for quantifying \"foci\" remains unclear and\nrequires a precise definition and a detailed description of the image analysis pipeline.\nResponse:\nWe thank the reviewer for this critical observation regarding the apparent\ndiscrepancy between our biochemical and cellular imaging data. We appreciate the\nopportunity to clarify this point.\nThe reviewer correctly notes that the in vitro EMSA assays demonstrate a clear\nreduction in binding affinity for the FMA-G4-2-Mut probe compared to the wild-type.\nThis result reflects the intrinsic, direct interaction affinity under purified conditions.\nIn the cellular environment, however, the interpretation of colocalization signals is\nmore complex. The fluorescent signal from the mutant probe in Fig. 2L likely\nrepresents a combination of factors, including potential low-affinity/background\nbinding, non-specific sequestration, or accumulation in cellular compartments, which\ncan be visualized by sensitive microscopy even when specific, high-affinity binding is\nsignificantly impaired. This is a known challenge in distinguishing specific from non-\nspecific interactions solely by colocalization microscopy. To strengthen our analysis\nand directly address the reviewer's concern, we have provided a detailed description\nof the image analysis pipeline in the Methods section in the revised manuscript,\nexplicitly defining how “foci” or “colocalization events” were identified and\nquantified. The quantification, based on this rigorous pipeline, confirms a statistically\nsignificant reduction in colocalization for the mutant probe compared to the wild-type,\nconsistent with the in vitro binding data. We apologize for any initial confusion\ncaused by the lack of methodological detail. We are grateful to the reviewer for\nprompting this essential clarification.\nThe description was added on page 8 of the revised supplementary information.\nThe discussion highlighted in yellow.\n4.Inadequate evidence for ZBP1-S oligomerization.\nThe evidence presented for ZBP1-S oligomerization is not convincing:\nFig. 3e, 3g (Split-GFP): The GFP signal is diffuse and does not form the discrete\npuncta or filaments expected for a template-driven oligomeric assembly. This pattern\ndoes not robustly support the conclusion of specific oligomerization.\nQuantification and definition:\nThe criteria for identifying an \"oligomer\" in the immunofluorescence images (Fig. 3J)\nare not defined. The quantification method must be transparently described.\nNeed for complementary evidence:\nThe claims of oligomerization require validation through more definitive biochemical and biophysical methods. Suggestions\ninclude Native PAGE, crosslinking, or SEC-MALS to determine oligomeric states.\nAdditionally, higher-resolution microscopy (e.g., SIM, Airyscan) would be necessary\nto properly visualize putative oligomeric structures.\nResponse:\nWe sincerely thank the reviewer for these critical and constructive suggestions\nregarding the evidence for ZBP1-S oligomerization. We fully agree with the assessment\nthat the initial split-GFP data, while indicative of interaction, does not provide\nconvincing proof of ZBP1-S oligomerization. Thus, as recommended, we performed\nsemi-denaturing detergent agarose gel electrophoresis (SDD-AGE), and found that\nTERRA could induce ZBP1-S oligomerization in the ZBP1-S overexpressed MCF-\nWT\n7 cells, but not TERRA (Fig. 3e). In addition, to directly visualize ZBP1-S\nMut\noligomerization in living cells, we performed Airyscan super-resolution microscopy.\nWe observed that upon induction of TERRA G4s, ZBP1-S forms distinct oligomeric\nfilaments (Fig. 3f). This provides compelling visual evidence for G4-driven ZBP1\nassembly. And we have added a detailed description of the definition and quantification\nof ZBP1 filaments (e.g., based on aspect ratio, length, and intensity profile), ensuring\nthe reproducibility of our analysis. We are grateful to the reviewer for this essential\nguidance, which has significantly strengthened this key aspect of our manuscript.\nThe added results were described on page 8 of the revised manuscript (Fig. 3e,f).\nThe description of the definition and quantification of ZBP1 filaments was added on\npage 8 and 9 of the revised supplementary information. The discussion highlighted in\nyellow.\nReviewer #2:\nThe revised manuscript by Qin et al. presents a substantially strengthened and\nwell-executed study demonstrating that human ZBP1 recognizes telomeric TERRA\nRNA through RNA G-quadruplex (G4) structures to activate MAVS-dependent innate\nimmune signaling. The authors have addressed all major concerns from the initial\nreview, and the new data provide robust mechanistic support for their conclusions.\nThe additional experiments, particularly the optimized EMSA and supershift assays\nconfirming direct and specific interaction between ZBP1-S and TERRA G4 RNA,\nthe inclusion of appropriate competition and non-G4 RNA controls, the validation of\nZBP1 knockout cells, and the expanded ligand-binding analysis testing the ability of\nZα domains to interact with multiple nucleic acid types (ssRNA, dsRNA,...),\ncollectively enhance the rigor of the study. The revised data provide convincing\nbiochemical and functional evidence that TERRA G4 structures are specifically\nrecognized by the Zα domain of ZBP1 and that this interaction triggers MAVS-\ndependent innate immune signaling and cell death. This finding is important because\nit expands the known spectrum of nucleic acid conformations sensed by ZBP1 beyond\ncanonical Z-form structures, establishing G4 RNA as a new class of innate immune\nligand and revealing a direct molecular link between telomeric RNA architecture and\nantiviral and inflammatory signaling.\nResponse:\nWe are deeply grateful to the reviewer for their exceptionally thoughtful and\npositive assessment of our work, and for their insightful suggestions which have\nsignificantly helped us in strengthening the manuscript. We are particularly encouraged\nby their recognition that our study \"provides important mechanistic insight\". We thank\nthe reviewer for the time spent on reviewing our manuscript, and the insightful\ncomments. The inputs and suggestions from the reviewer have been implemented in\nthe manuscript. Please see our point-to-point response below.\n1. The authors clarify that fluorescence complementation in the split GFP (BiFC)\nsystem reflects the proximity of tagged ZBP1 molecules and cite previous studies using\nthis approach to infer oligomerization. While this rationale is understood, the method\nintrinsically cannot distinguish between direct ZBP1–ZBP1 interactions and the co-\nlocalization of multiple ZBP1 molecules on the same multimeric or stacked G4 RNA\nscaffold. Although the BiFC data are consistent with ligand-induced proximity of ZBP1\nmolecules, they do not by themselves demonstrate bona fide oligomerization. To\nstrengthen this conclusion, the authors could examine ZBP1 under RNA-free conditions using non-reducing, non-denaturing\nnative PAGE, which preserves weak intermolecular contacts and can reveal distinct higher molecular weight assemblies\nrelative to the monomer. Native PAGE has been successfully used to demonstrate\noligomerization of other nucleic acid sensors and adaptors, including MAVS, and could\nsimilarly determine whether ZBP1 forms stable higher order assemblies. This analysis\nwould fill a key gap in the current study by providing direct biochemical evidence for\nZBP1 self-association.\nResponse:\nWe sincerely thank the reviewer for these critical and constructive suggestions\nregarding the evidence for ZBP1-S oligomerization. We fully agree with the assessment\nthat the initial split-GFP data, while indicative of interaction, does not provide\nconvincing proof of ZBP1-S oligomerization. Thus, as recommended, we performed\nsemi-denaturing detergent agarose gel electrophoresis (SDD-AGE), and found that\nTERRA could induce ZBP1-S oligomerization in the ZBP1-S overexpressed MCF-\nWT\n7 cells, but not TERRA (Fig. 3e). We are grateful to the reviewer for this excellent\nMut\nguidance, which has significantly strengthened the mechanistic rigor of our study.\nThe added results were described on page 8 of the revised manuscript (Fig. 3e).\nThe discussion highlighted in yellow.\n2. The cell viability data currently presented in Extended Data Fig. 3 are essential for\ninterpreting the functional consequences of ZBP1 activation and should be quantified\nand incorporated into the main Figure 3. In addition, the image quality in Figure 3f is\npoor, and the visualization of filament-like ZBP1 structures is not convincing. The\nauthors should improve imaging resolution and include quantitative analysis of the\nproportion of cells exhibiting filamentous ZBP1 structures in Figure 3 to substantiate\nthis observation.\nResponse:\nWe thank the reviewer for these essential suggestions regarding data presentation\nand image quality, which have significantly improved the clarity and impact of our\nfindings. As suggested, we have now quantified the cell viability data from Extended\nData Fig. 3 and incorporated it as a new panel (Fig. 3h,i) in the main figure. This\ndirectly integrates the functional consequence of ZBP1 activation into the core\nnarrative of the study. Furthermore, we fully agree that the original image in Fig. 3f\nwas insufficient to convincingly demonstrate filamentous structures. In response, we\nhave replaced this panel with new, high-resolution images acquired using Airyscan\nsuper-resolution microscopy. These new images clearly reveal the elongated,\nfilament-like oligomers of ZBP1-S induced by TERRA G4 (Fig. 3f). Moreover, we\nhave added a quantitative analysis that reports the ZBP1-S filaments number per cell\n(Fig. 3f). We believe these revisions have fully addressed the reviewer's concerns,\nsubstantially strengthening both the visual evidence and the quantitative rigor of Fig.\n3.We are grateful for these constructive comments.\nThe added results were described on page 8 of the revised manuscript (Fig. 3f).\nThe discussion highlighted in yellow.\nReviewer #3:\nAfter reading the revised manuscript and the responses to all reviewers, I\nrecommend the publication of the work in Nat. Commun.\nResponse:\nWe are profoundly grateful for your final assessment and for your\nrecommendation to publish our manuscript in Nature Communications. We sincerely\nthank you for the considerable time, expertise, and thoughtful guidance you have\ndedicated throughout the review process. Your insightful comments and constructive\ncritiques have been instrumental in significantly strengthening the quality, clarity, and\nrigor of our work. We deeply appreciate your role in shaping this study and are\nhonored by your endorsement."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I believe the authors have addressed most of my concerns and I congratulate them for this work.", "reviewer_2": "My previous concerns regarding the evidence for a ZBP1(S)–TERRA interaction and ligand-induced ZBP1 oligomerization\nhave been fully addressed in the revised manuscript. The new data convincingly demonstrate that the ZBP1–TERRA\nassociation is direct, and they provide biochemical evidence supporting ZBP1 oligomer formation in response to TERRA G-\nquadruplex (G4) RNA. The additional experiments reinforce the mechanistic rigor of the work and further substantiate its\ncentral conclusions.\nMinor points:\n1.A key mechanistic advance in this work is the finding that TERRA G4 RNA binding via the Zα domain promotes ZBP1\noligomerization. This aspect is both novel and should be highlighted more explicitly in (i) the abstract and (ii) the discussion.\nIn the discussion, it would also be valuable to propose a working model illustrating how nucleic acid engagement drives\nhigher-order ZBP1 assembly, and to frame this within the broader context of RNA-templated oligomerization mechanisms\nestablished for RIG-I-like receptors (RLRs), particularly RIG-I and MDA5. RIG-I (DDX58) and MDA5 (IFIH1) represent\nparadigmatic examples of innate immune sensors in which ligand binding is coupled to cooperative oligomerization to\nachieve a signaling-competent state. This should be discussed more explicitly.\n2.In Figure 2F, the labeling of the last two lanes appears to be identical; please verify and correct if necessary."}, "author_response": "Next is the summarization of our point-by-point response to the comments raised by\nthe reviewers.\nTo Reviewer #1:\nI believe the authors have addressed most of my concerns and I congratulate\nthem for this work.\nResponse:\nWe are profoundly grateful for your final assessment and for your\nrecommendation to publish our manuscript in Nature Communications. We sincerely\nthank you for the considerable time, expertise, and thoughtful guidance you have\ndedicated throughout the review process. Your insightful comments and constructive\ncritiques have been instrumental in significantly strengthening the quality, clarity, and\nrigor of our work. We deeply appreciate your role in shaping this study and are\nhonored by your endorsement.\nTo Reviewer #2:\nMy previous concerns regarding the evidence for a ZBP1(S)–TERRA interaction and\nligand-induced ZBP1 oligomerization have been fully addressed in the revised\nmanuscript. The new data convincingly demonstrate that the ZBP1–TERRA\nassociation is direct, and they provide biochemical evidence supporting ZBP1 oligomer\nformation in response to TERRA G-quadruplex (G4) RNA. The additional experiments\nreinforce the mechanistic rigor of the work and further substantiate its central\nconclusions.\nResponse:\nWe are deeply grateful to the reviewer for their exceptionally thoughtful and\npositive assessment of our work, and for their insightful suggestions which have\nsignificantly helped us in strengthening the manuscript. We are particularly encouraged\nby their recognition that our study \"provides important mechanistic insight\". We thank\nthe reviewer for the time spent on reviewing our manuscript, and the insightful\ncomments. The inputs and suggestions from the reviewer have been implemented in\nthe manuscript. Please see our point-to-point response below.\n1.A key mechanistic advance in this work is the finding that TERRA G4 RNA binding\nvia the Zα domain promotes ZBP1 oligomerization. This aspect is both novel and\nshould be highlighted more explicitly in (i) the abstract and (ii) the discussion. In the\ndiscussion, it would also be valuable to propose a working model illustrating how\nnucleic acid engagement drives higher-order ZBP1 assembly, and to frame this within\nthe broader context of RNA-templated oligomerization mechanisms established for\nRIG-I-like receptors (RLRs), particularly RIG-I and MDA5. RIG-I (DDX58) and\nMDA5 (IFIH1) represent paradigmatic examples of innate immune sensors in which\nligand binding is coupled to cooperative oligomerization to achieve a signaling-\ncompetent state. This should be discussed more explicitly.\nResponse:\nWe sincerely thank the reviewer for their insightful and constructive comment.\nWe agree entirely that highlighting the mechanistic link between TERRA G4 binding\nand ZBP1 oligomerization is crucial for emphasizing the novelty and significance of\nour findings. As suggested, we have now explicitly addressed this point in both the\nabstract and the discussion section. Thank you again for this valuable suggestion.\n2. In Figure 2F, the labeling of the last two lanes appears to be identical; please verify\nand correct if necessary.\nResponse:\nWe sincerely thank the reviewer for their careful attention to detail. The reviewer\nis absolutely correct regarding the labeling error in the original Figure 2F. The labeling\nof the last two lanes was indeed identical due to a clerical mistake. In the revised\nmanuscript, we have corrected this error. The figure legend and the labeling in the\nfigure file itself have been updated to accurately reflect the experimental conditions for\nall lanes. We greatly appreciate the reviewer pointing this out, as it ensures the clarity\nand accuracy of our data presentation. Thank you again for this valuable correction."}]

Decision letter