Biology subjects

Geyer, M.

Publications and source records attributed to Geyer, M..

At least 19 recordsLinked to original sources

bioRxiv2026

NLRP3 acts as a direct sensor of intracellular potassium ions

The NLRP3 inflammasome is a sentinel of cellular homeostasis, and its activation triggers the assembly of a molecular machinery that drives inflammation in infection, cardiovascular, metabolic, and neurodegenerative diseases. The majority of the many triggers known to activate NLRP3 are believed to induce potassium ion (K+) efflux from the cell as a fundamental danger signal for compromised cellular integrity. However, it has remained unclear how a reduction in intracellular K+ concentration is mechanistically translated into conformational changes in NLRP3 that promote inflammasome assembly, interleukin (IL)-1 release, and cell death. Here, we provide evidence that alterations in K+ levels directly regulate the conformation of the NLRP3 protein. In cell-free lysates derived from cell lines and primary blood immune cells high K+ concentrations stabilized a compact, protease-resistant structure resembling inhibitor-bound NLRP3, whereas low K+ conditions or the presence of a K+ chelator favored an open, more flexible and protease-accessible conformation. Notably, human NLRP3 remained responsive to K+ even when exogenously expressed in macrophage-like Drosophila cells or purified as recombinant protein. This indicates that K+ sensing occurs independently of cellular co-factors and is consistent with direct ion coordination. Of note, stimulation with the K+-independent NLRP3 agonist CL097 failed to recapitulate the conformational transition caused by K+ efflux inducer, nigericin. Moreover, pathogenic gain-of-function mutant variants of NLRP3 constitutively resembled the open and flexible protease-accessible conformation. Mapping K+-interactions by high-performance computation suggested that K+ ions populate the nucleotide binding pocket of the FISNA-NACHT module of individual NLRP3 chains but also stabilize face-to-face interactions within inactive oligomeric cage assemblies via the NACHT-adjacent acidic loop. Collectively, our findings enable us to propose a mechanistic model of how intracellular K+ ions preclude NLRP3 activation prior to efflux and thus how NLRP3 responds to cellular danger as a direct K+ sensing protein.

immunology↗

bioRxiv2026

Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

Recent advances in AI-based structural biology have made de novo protein binder design increasingly effective, yet performance remains highly target dependent. For instance, the cancer surface antigen Nectin-4, an immunoglobulin-like cell adhesion protein, proved particularly challenging for RFdiffusion-based minibinder generation, yielding substantially fewer high-quality candidates than related targets. To address this bottleneck, we integrated an evolutionary genetic algorithm (GA) with AI-driven design. GA selection with tunable stringency was coupled with diversification via partial diffusion or direct sequence editing, enabling efficient exploration of sequence-structure space and rapid enrichment of promising candidates. This AI-GA pipeline quickly produced large and diverse minibinder panels with very good in silico quality metrics and is compatible with inputs from multiple design algorithms. Pooled, large-scale experimental screening identified highly stable Nectin-4 minibinders with single-digit nanomolar down to subnanomolar affinities. Lead binders were further engineered into Nectin-4-specific flow cytometry detection reagents and potent bispecific T cell engagers, demonstrating functional activity beyond binding. Together, these results show that evolutionary refinement can unlock challenging targets and accelerate de novo protein design for next-generation cancer biologics.

bioengineering↗

bioRxiv2025

Quantitative modeling of P-TEFb mediated CTD phosphorylation identifies local cooperativity

Fine-tuned regulation of RNA polymerase II (Pol II) activity is essential for accurate gene expression. A key layer of this regulation involves phosphorylation of Pol IIs C-terminal domain (CTD), a repetitive heptapeptide tail that coordinates transcription and RNA-processing factors. The kinase P-TEFb plays a major role in this process, yet its precise phosphorylation mechanism remains unclear. Previous in vitro studies have suggested a distributive mode of action based largely on qualitative inspection of mass spectrometry data rather than quantitative analysis. Here, we use mathematical modeling of CTD phosphorylation to explore whether local context, such as neighboring phosphorylations or directional biases, affects PTEFb activity on the CTD. Our results indicate that P-TEFb acts distributively but with pronounced local cooperativity: repeats adjacent to phosphorylated sites are modified at higher rates. We find no evidence for directional bias, although the limited positional resolution of the data precludes a definitive conclusion. These results identify local context as an important factor in P-TEFb-mediated CTD phosphorylation and establish a quantitative modeling framework for dissecting multi-site modification dynamics.

systems biology↗

bioRxiv2025

C-terminal lysine residues localise NLRP10 at lipid compartments and govern NLRP10 oligomer formation

NLRP10 is an atypical member of the NOD-like receptor (NLR) family because it lacks a leucine-rich repeat domain at its C-terminus. Recently, NLRP10 has been shown to form an inflammasome, however, the mechanism and the trigger of activation remain elusive. Here, we show that in human epithelial cells and keratinocytes NLRP10 oligomerises in response to the stressor m-3M3FBS. NLRP10 co-localises with ASC upon overexpression, but ASC nucleation and recruitment were different to other NLRPs. While neither ATP hydrolysis nor the pyrin domain were required, the C-terminal tail region (aa 584-655) was both necessary and sufficient for oligomerisation. Generation of chimeric proteins showed that the function of the tail is conserved between human and mouse NLRP10 in respect to aggregation but convers different protein stability. Changes in the subcellular localisation of NLRP10 and oligomerization were dependent on the presence of evolutionarily conserved lysine residues in the tail region, which localise the majority of NLRP10 at lipid droplets. Our study identifies the C-terminal basic tail of NLRP10 as a key regulatory element for oligomerisation and localisation at lipid interfaces. These findings underline differences of NLRP10 activation in respect to other inflammasome forming NLRPs and suggest a role of lipids in NLRP10 activation.

immunology↗

bioRxiv2025

Inhibition of NLRP3 by a CNS-penetrating indazole scaffold

Low-grade inflammation is a hallmark of ageing and a key cause of age-related impairments and diseases1. The NOD-like receptor NLRP3 senses a variety of danger signals and environmental insults, resulting in pro-inflammatory response, inflammasome formation and pyroptosis2,3. Its aberrant activation has been linked to many acute and chronic diseases ranging from atherosclerosis to Alzheimers disease and cancer, making NLRP3 an attractive therapeutic target4,5. Here we report the discovery, characterization, and structure of an indazole-based NLRP3 antagonist, BAL-1516, which potently inhibits inflammasome formation in monocytes and microglia. The cryo-electron microscopy structure of BAL-1516 bound to NLRP3 reveals a previously undescribed compound binding site at a surface groove of the nucleotide-binding domain with contacts to the FISNA and WHD subdomains. The characteristic feature of BAL compound binding is the formation of three hydrogen bonds to the peripheral {beta}-strand of the triple-ATPase; two from the indazoles nitrogen atoms and a third from the compounds linker region. Additional phenyl and thiazole moieties render the compound hydrophobic, allowing excellent blood-brain barrier penetration. The compound binding site is highly specific for NOD-like receptors, and the optimized compound BAL-1516 is able to directly bind mouse NLRP3 despite two conservative residue changes in the binding interface. The BAL compounds represent a first-in-class family of NLRP3 inhibitors, providing a broad design space, including covalent and degradative properties, for the development of NLRP3-directed therapeutics. The innate immune system contains cytosolic proteins that sense cellular stress caused by bacterial, viral and fungal infections or sterile inflammation, to control cellular integrity2. NLRP3 is a well-studied member of the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) that is involved in the activation of the inflammasome, a multiprotein complex that mediates inflammation6. Upon detection of stress or pathogen-associated signals, NLRP3 triggers the activation of caspase-1, which leads to the production of pro-inflammatory cytokines such as IL-1{beta} and IL-18, driving inflammatory responses and ultimately pyroptotic cell death. In the context of neuroinflammation, NLRP3 plays a significant role in the pathogenesis of various neurodegenerative diseases, including Alzheimers disease, Parkinsons disease, and multiple sclerosis7. Research into targeting NLRP3 signalling with CNS-penetrating molecules holds potential for developing therapeutic strategies to alleviate neuroinflammatory conditions and to slow the progression of neurodegenerative diseases.

immunology↗

bioRxiv2025

Development of AI-designed protein binders for detection and targeting of cancer cell surface proteins

Artificial intelligence (AI)-based protein design opens new avenues for the rapid generation of new research tools and therapeutics, but experimental validation lags behind the computational design throughput. Here, we present a scalable workflow for the discovery and validation of AI-designed protein binders. Leveraging the RFdiffusion protein design pipeline with a custom filter for stable alpha-helical bundle folds, we construct libraries of thousands of AI-binders against cancer-associated surface proteins. Mammalian cell-surface and phage display screening yield multiple high-affinity PD-L1 binders but fewer hits for CD276 (B7-H3) and VTCN1 (B7-H4), reflecting the target-dependent efficiency of RFdiffusion in generating high-quality designs. Using our experimentally validated AI-designed binder libraries, we benchmark freely available structure prediction models. We find that interface predicted template modelling (ipTM) scores by Chai-1 with ESM embedding correlate well with experimental success and even predict deleterious effects of binding interface mutations. To demonstrate the versatility of AI-binders as research tools, we deploy them in CAR-T cells and also assemble them with fluorophore-labeled streptavidin into tetravalent quattrobinders, which achieve antibody-comparable staining of endogenous PD-L1 by flow cytometry. With high production yields and accessible structural models, AI-designed quattrobinders are versatile and cost-effective research tools amenable to community-driven validation and optimization.

bioengineering↗

bioRxiv2025

The MYCN/Aurora-A complex is a cyclin activating kinase for CDK12

Deregulated MYCN is a driver of aggressive pediatric and adult neuroendocrine tumors, but critical oncogenic processes downstream of MYCN remain poorly defined. In neuroblastoma, MYCN interacts with and activates the Aurora-A kinase. Here we show that Aurora-A is a CDK-activating kinase for CDK12 by phosphorylating T893 in the T-loop, thereby enhancing its kinase activity. Aurora-A-dependent activation of CDK12 controls phosphorylation of T4 of RNA polymerase and recruits transcription termination complexes, thereby preventing transcription-replication conflicts. Enhanced crosslinking and immunoprecipitation sequencing reveals that Aurora-A associates with splice sites on nascent RNA. RNA-bound Aurora-A is catalytically inactive. MYCN competes with RNA for binding to Aurora-A and displaces Aurora-A from RNA in cells, promoting its CDK12 kinase activity. Combining Aurora-A and CDK12 inhibition potently suppresses the growth of MYCN-amplified neuroblastoma cells and patient-derived xenografts. Our data demonstrate that an Aurora-A/CDK12-dependent transcription termination pathway is a critical and targetable dependency of MYCN-driven tumors.

cancer biology↗

bioRxiv2024

Discovery of a Potent and Selective Inhibitor of Human NLRP3 with a Novel Binding Modality and Mechanism of Action

The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1{beta} and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA encoded library screen to identify novel NLRP3 binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950 binding pocket. Using humanized NLRP3 mice we show that a derivative of BAL-0028 inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that BAL-0028 inhibits select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than does MCC950. BAL-0028 thus represents a new modality for NLRP3 inhibition in inflammatory diseases. SUMMARYNLRP3 is a target for anti-inflammatory therapies and can be inhibited by the tool compound MCC950. We describe the characterization of a new small molecule inhibitor of NLRP3 BAL-0028 that has a distinct mechanism of action and binding site.

immunology↗

bioRxiv2024

The inflammasome sensor NLRP3 interacts with REV7 to maintain genome integrity through homologous recombination

DNA double strand break (DSB) is a highly toxic lesion that can generate genome instability, a major source of tumorigenesis. DSBs are mainly repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The selection of the DSB repair pathway primarily depends on the DNA resection of the DSB ends. Indeed, HR is initiated by resection at the DSB generating 3 single stranded extension. The shieldin complex prevents resection fostering DSB repair toward NHEJ. Here, we reveal that the inflammasome sensor NLRP3 facilitates DNA end resection to promote the HR pathway in an inflammasome-independent manner. Strikingly, NLRP3 silencing decreases HR efficiency, as evidenced by RAD51 foci and functional HR assays. Mechanistically, we describe that NLRP3 interacts with REV7, a subunit of the shieldin complex, and its depletion increases REV7 recruitment to IR-induced DSBs. Similar to cancer cells harboring HR mutated genes, we find that NLRP3 deficient cells are sensitive to PARP inhibitors (PARPi) and exhibit an epistatic relationship with BRCA1 deficiency. Remarkably, loss of REV7 in NLRP3-depleted cells induces PARPi resistance by restoring HR. This study unravels the crucial role of the innate immune receptor NLRP3 in regulating the selection of DSB repair pathways to maintain genome integrity.

molecular biology↗

bioRxiv2024

The SAVED domain of the type III CRISPR protease CalpL is a ring nuclease

Prokaryotic CRISPR-Cas immune systems detect and cleave foreign nucleic acids. In type III CRISPR-Cas systems, the Cas10 subunit of the activated recognition complex synthesizes cyclic oligoadenylates (cOAs), second messengers that activate downstream ancillary effector proteins. Once the viral attack has been weathered, elimination of extant cOA is essential to limit the antiviral response and to allow cellular recovery. Various families of ring nucleases have been identified, specializing in the degradation of cOAs either as standalone enzymes or as domains of effector proteins. Here we describe the ring nuclease activity inherent in the SAVED domain of the cA4-activated CRISPR Lon protease CalpL. We characterize the kinetics of cA4 cleavage and identify key catalytic residues. We demonstrate that cA4-incuced oligomerization of CalpL is essential not only for activation of the protease, but is also required for nuclease activity. Further, the nuclease activity of CalpL poses a limitation to the protease reaction, indicating a mechanism for regulation of the CalpL/T/S signaling cascade. This work is the first demonstration of a catalytic SAVED domain and gives new insights into the dynamics of transcriptional adaption in CRISPR defense systems which are not aimed at abortive infection but rather at a reversible adaption to phage attack.

biochemistry↗

bioRxiv2024

Structural basis of Cdk7 activation by dual T-loop phosphorylation

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates--the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

biochemistry↗

bioRxiv2023

Functional diversity of NLRP3 gain-of-function mutants associated with CAPS autoinflammation.

NLRP3-associated autoinflammatory disease (NLRP3-AID or CAPS) is an heterogenous group of monogenic autoinflammations associated with NLRP3 gain-of-function mutations. The poor functional characterization of most NLRP3 variants is a barrier to diagnosis although patients can be efficiently treated with anti-IL-1 approaches. In addition, while NLRP3 inflammasome is controlled by coordinated priming and activation signals, gain-of-functions of NLRP3 variants have been only investigated in response to priming. Here, we functionally characterize 34 NLRP3 variants in vitro by determining their activity in response to induction, priming and/or activation signals, and their sensitivity to inhibitors. We highlight the functional diversity of the gain-of-function mutants and describe four groups based on their profile of signals required for their activation, that correlate partly with the symptoms severities. We identify a new group of NLRP3 mutants responding to the activation signal without priming, with patients often misdiagnosed. Our results identify key NLRP3 residues controlling the inflammasome activity and sensitivity to inhibitors. The comparison of four inhibitors on the 34 variants identifies inhibitory mechanisms with broader efficiency for future drug design. Altogether, our results provide new insights on NLRP3 activation and an explanatory mechanism for NLRP3-AID heterogeneity, and original tools for NLRP3-AID diagnosis and anti-inflammatory disease drug development. eTOC SummaryFunctional characterization of 34 CAPS-associated NLRP3 variants identifies polymorphisms versus gain-of-function pathogenic mutants, and highlights diversities in the signals controlling their activation and in their sensitivity to inhibitors. This study provides tools for CAPS diagnosis and anti-inflammation drug development and insights on NLRP3 control mechanisms.

immunology↗

bioRxiv2023

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b (P-TEFb) required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9 and is not involved with releasing P-TEFb from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

cell biology↗

bioRxiv2023

Non-decameric NLRP3 forms an MTOC-independent inflammasome

NLRP3 is an inflammasome-forming protein that plays a key role in conditions ranging from infections to Alzheimers disease. NLRP3 is activated by many potassium (K+)-dependent or - independent stimuli related to pathogens or sterile insults. Molecularly, human NLRP3 assembles into a decameric cage and its interaction with both the trans-Golgi network (TGN) and the microtubule organization centre (MTOC) has been proposed as critical for NLRP3 activation. However, the relative mechanistic requirement for K+-dependent versus - independent stimuli is unclear. Using spatially and dynamically high-resolution microscopy in human macrophages, we found that K+-dependent NLRP3 stimulation triggers two distinct activation pathways: (i) the decameric cage-dependent pathway, which was also exclusively required for the K+-independent stimulus, imiquimod; and (ii) a cage- and TGN/MTOC-independent pathway that was fully functional in an NLRP3 protein engineered to form monomers and unable to interact with membrane lipids. Collectively, our results delineate two parallel yet biologically distinct NLRP3 activation pathways.

molecular biology↗

bioRxiv2023

hnRNPM and ELAVL1 control type I interferon induction by promoting IRF3 phosphorylation downstream of both cGAS and RIG-I

RIG-I and cGAS are crucial sensors of viral nucleic acids and induce type I IFNs via TBK1/IKK and IRF3. Here, we have identified hnRNPM as a novel positive regulator of IRF3 phosphorylation and type I IFN induction downstream of both cGAS and RIG-I. Combining interactome analysis and genome editing, we further identified ELAVL1 as an immune-relevant interactor of hnRNPM. Depletion of hnRNPM or ELAVL1 impaired type I IFN induction by HSV-1 and SeV. In addition, we found that hnRNPM and ELAVL1 interact with TBK1 and NF-kB p65. Confocal microscopy revealed cytosolic and perinuclear interactions between hnRNPM, ELAVL1, and TBK1. To our knowledge, hnRNPM and ELAVL1 represent the first non-redundant signaling components merging the cGAS-STING and RIG-I-MAVS pathways, thus representing a novel platform that fuels antiviral defense.

immunology↗

bioRxiv2023

Antagonistic nanobodies reveal mechanism of GSDMD pore formation and unexpected therapeutic potential

Activation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. Yet, it remained unclear what triggers the conformational changes that allow membrane insertion, as methods to study pore formation in living cells were limited. We raised nanobodies specific for human GSDMD and found two nanobodies that prevent pyroptosis and IL-1{beta} release when expressed in the cytosol of human macrophages. Nanobody binding to GSDMDNT blocked its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNT partitioned into the plasma membrane, suggesting that pore formation is initiated by insertion of monomers, followed by oligomerization in the target membrane. When GSDMD pore formation was inhibited, cells still underwent caspase-1-dependent apoptosis, likely due to the substantially augmented caspase-1 activity. This hints at a novel layer of regulation of caspase-1 activity by GSDMD pores. Moreover, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the medium prevented cell death by pyroptosis, likely by entering through GSDMD pores and curtailing the assembly of additional pores. GSDMD nanobodies may thus be suitable to treat the ever-growing list of diseases caused by activation of the (non-) canonical inflammasomes.

immunology↗

bioRxiv2023

Pyroptosis inhibiting nanobodies block Gasdermin D pore formation

Gasdermin D (GSDMD) is a key mediator of pyroptosis, a pro-inflammatory form of cell death occurring downstream of inflammasome activation as part of the innate immune defence. Upon cleavage by inflammatory caspases, the N-terminal domain of GSDMD forms pores in the plasma membrane resulting in cytokine release and eventually cell death. Targeting GSDMD is an attractive way to dampen inflammation. In this study, six GSDMD targeting nanobodies were characterized in terms of their binding affinity, stability, and effect on GSDMD pore formation. Three of the nanobodies inhibited GSDMD pore formation in a liposome leakage assay, although caspase cleavage was not perturbed. We determined the crystal structure of human GSDMD in complex with two nanobodies at 1.9 [A] resolution, providing detailed insights into the GSDMD-nanobody interactions and epitope binding. The pore formation is sterically blocked by one of the nanobodies that binds to the oligomerization interface of the N-terminal domain in the multi-subunit assembly. Our biochemical and structural findings provide new tools for studying inflammasome biology and build a framework for the design of novel GSDMD targeting drugs.

biochemistry↗

bioRxiv2022

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5triphosphorylated and 5base-paired RNA(dsRNA). Here, we found that, while 5unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5-monophosphate(5p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with a long, highly structured, polyA-bearing, non-coding mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5p-RNA recognition is crucial to preventing mtRNA-triggered RIG-I-mediated autoinflammation.

immunology↗