Recognition Of Double-stranded RNA Research Articles

Structural basis for double-stranded RNA recognition by SID1.

The nucleic acid transport properties of the systemic RNAi-defective (SID) 1 family make them attractive targets for developing RNA-based therapeutics and drugs. However, the molecular basis for double-stranded (ds) RNA recognition by SID1 family remains elusive. Here, we report the cryo-EM structures of Caenorhabditis elegans (c) SID1 alone and in complex with dsRNA, both at a resolution of 2.2 Å. The dimeric cSID1 interacts with two dsRNA molecules simultaneously. The dsRNA is located at the interface between β-strand rich domain (BRD)1 and BRD2 and nearly parallel to the membrane plane. In addition to extensive ionic interactions between basic residues and phosphate backbone, several hydrogen bonds are formed between 2'-hydroxyl group of dsRNA and the contact residues. Additionally, the electrostatic potential surface shows three basic regions are fitted perfectly into three major grooves of dsRNA. These structural characteristics enable cSID1 to bind dsRNA in a sequence-independent manner and to distinguish between DNA and RNA. The cSID1 exhibits no conformational changes upon binding dsRNA, with the exception of a few binding surfaces. Structural mapping of dozens of loss-of-function mutations allows potential interpretation of their diverse functional mechanisms. Our study marks an important step toward mechanistic understanding of the SID1 family-mediated dsRNA uptake.

Structural insights into double-stranded RNA recognition and transport by SID-1.

RNA uptake by cells is critical for RNA-mediated gene interference (RNAi) and RNA-based therapeutics. In Caenorhabditis elegans, RNAi is systemic as a result of SID-1-mediated double-stranded RNA (dsRNA) across cells. Despite the functional importance, the underlying mechanisms of dsRNA internalization by SID-1 remain elusive. Here we describe cryogenic electron microscopy structures of SID-1, SID-1-dsRNA complex and human SID-1 homologs SIDT1 and SIDT2, elucidating the structural basis of dsRNA recognition and import by SID-1. The homodimeric SID-1 homologs share conserved architecture, but only SID-1 possesses the molecular determinants within its extracellular domains for distinguishing dsRNA from single-stranded RNA and DNA. We show that the removal of the long intracellular loop between transmembrane helix 1 and 2 attenuates dsRNA uptake and systemic RNAi in vivo, suggesting a possible endocytic mechanism of SID-1-mediated dsRNA internalization. Our study provides mechanistic insights into dsRNA internalization by SID-1, which may facilitate the development of dsRNA applications based on SID-1.

Mitochondrial Reactive Oxygen Species in TRIF-Dependent Toll-like Receptor 3 Signaling in Bronchial Epithelial Cells against Viral Infection.

Toll-like receptor 3 (TLR3) plays an important role in double-stranded RNA recognition and triggers the innate immune response by acting as a key receptor against viral infections. Intracellular reactive oxygen species (ROS) are involved in TLR3-induced inflammatory responses during viral infections; however, their relationship with mitochondrial ROS (mtROS) remains largely unknown. In this study, we show that polyinosinic-polycytidylic acid (poly(I:C)), a mimic of viral RNA, induced TLR3-mediated nuclear factor-kappa B (NF-κB) signaling pathway activation and enhanced mtROS generation, leading to inflammatory cytokine production. TLR3-targeted small interfering RNA (siRNA) and Mito-TEMPO inhibited inflammatory cytokine production in poly(I:C)-treated BEAS-2B cells. Poly(I:C) recruited the TLR3 adaptor molecule Toll/IL-1R domain-containing adaptor, inducing IFN (TRIF) and activated NF-κB signaling. Additionally, TLR3-induced mtROS generation suppression and siRNA-mediated TRIF downregulation attenuated mitochondrial antiviral signaling protein (MAVS) degradation. Our findings provide insights into the TLR3-TRIF signaling pathway and MAVS in viral infections, and suggest TLR3-mtROS as a therapeutic target for the treatment of airway inflammatory and viral infectious diseases.

Novel insights into double-stranded RNA-mediated immunopathology.

Recent progress in human and mouse genetics has transformed our understanding of the molecular mechanisms by which recognition of self double-stranded RNA (self-dsRNA) causes immunopathology. Novel mouse models recapitulate loss-of-function mutations in the RNA editing enzyme ADAR1 that are found in patients with Aicardi-Goutières syndrome (AGS) - a monogenic inflammatory disease associated with increased levels of type I interferon. Extensive analyses of the genotype-phenotype relationships in these mice have now firmly established a causal relationship between increased intracellular concentrations of endogenous immunostimulatory dsRNA and type I interferon-driven immunopathology. Activation of the dsRNA-specific immune sensor MDA5 perpetuates the overproduction of type I interferons, and chronic engagement of the interferon-inducible innate immune receptors PKR and ZBP1 by dsRNA drives immunopathology by activating an integrated stress response or by inducing excessive cell death. Biochemical and genetic data support a role for the p150 isoform of ADAR1 in the cytosol in suppressing the spontaneous, pathological response to self-dsRNA.

The RNA-binding protein ZFP36 strengthens innate antiviral signaling by targeting RIG-I for K63-linked ubiquitination.

Innate immunity is the first line of defense against infections, which functions as a significant role in resisting pathogen invasion. Rapid immune response is initiated by pattern recognition receptors (PRRs) quickly distinguishing "self" and "non-self." Upon evolutionarily conserved pathogen-associated molecular pattern (PAMP) is recognized by PRRs, innate immune response against infection is triggered via an orchestration of molecular interaction, cytokines cascades, and immune cells. RIG-I plays a critical role in type I interferon (IFN-I) production by direct recognition of cytoplasmic double-stranded viral RNA. However, the activation mechanism of RIG-I is incompletely understood. In this study, we reported RNA-binding protein ZFP36 as a positive regulator of RIG-I-mediated IFN-I production. ZFP36 is a member of Zinc finger proteins (ZFPs) characterized by the zinc finger (ZnF) motif, which broadly involved gene transcription and signal transduction. However, its role in regulating antiviral innate immune signaling is still unclear. We found that ZFP36 associates with RIG-I and potentiates the FN-β production induced by SeV. Mechanistically, ZFP36 promotes K63-linked polyubiquitination of RIG-I, mostly at K154/K164/K172, thereby facilitating the activation of RIG-I during infection. While the mutant ZFP36 (C118S/C162S) failed to increase polyubiquitination of RIG-I and SeV induced FN-β. Our findings collectively demonstrated that ZFP36 acts as a positive regulator in antiviral innate immunity by targeting RIG-I for K63-linked ubiquitination, thus improving our understanding of the activation mechanism of RIG-I.

Translation Arrest: A Key Player in Plant Antiviral Response.

Plants evolved several mechanisms to protect themselves against viruses. Besides recessive resistance, where compatible host factors required for viral proliferation are absent or incompatible, there are (at least) two types of inducible antiviral immunity: RNA silencing (RNAi) and immune responses mounted upon activation of nucleotide-binding domain leucine-rich repeat (NLR) receptors. RNAi is associated with viral symptom recovery through translational repression and transcript degradation following recognition of viral double-stranded RNA produced during infection. NLR-mediated immunity is induced upon (in)direct recognition of a viral protein by an NLR receptor, triggering either a hypersensitive response (HR) or an extreme resistance response (ER). During ER, host cell death is not apparent, and it has been proposed that this resistance is mediated by a translational arrest (TA) of viral transcripts. Recent research indicates that translational repression plays a crucial role in plant antiviral resistance. This paper reviews current knowledge on viral translational repression during viral recovery and NLR-mediated immunity. Our findings are summarized in a model detailing the pathways and processes leading to translational arrest of plant viruses. This model can serve as a framework to formulate hypotheses on how TA halts viral replication, inspiring new leads for the development of antiviral resistance in crops.

TLR3 recognition of viral double-stranded RNA in human dental pulp cells is important for the innate immunity

Dental caries or trauma can expose human dental pulp cells (DPCs) to various oral microorganisms, which play an important role in the development of an innate immune response. In the present study, we examined the expression of Toll-like receptors (TLRs) for sensing microbe-associated molecular patterns in human DPCs. Interestingly, real-time PCR analysis demonstrated that TLR3 is the most highly expressed among 10 different TLRs in human DPCs. Poly(I:C), a representative TLR3 ligand mimicking viral double-stranded RNA, potently induced IL-8 expression in a time- and dose-dependent manner. Concordantly, poly(I:C) treatment substantially increased the expression of pro-inflammatory cytokines and chemokines such as IL-6, CCL2, and CXCL10. Human DPCs transfected with TLR3 siRNA exhibited decreased IL-8 production compared with non-targeting siRNA-transfected cells, suggesting that the expression of poly(I:C)-induced inflammatory cytokines is dependent on TLR3. IL-8 secretion induced by poly(I:C) was down-regulated by MAP kinase inhibitors, indicating that the MAP kinase pathway contributes to IL-8 production. Furthermore, C/EBPβ and NF-κB were essential transcriptional factors for poly(I:C)-induced IL-8 expression, as demonstrated by the transient transfection and reporter gene assay. Since lipoproteins are known as major immunostimulatory components of bacteria, human DPCs were treated with poly(I:C) together with Pam2CSK4, a synthetic lipopeptide mimicking bacterial lipoproteins. Pam2CSK4 and poly(I:C) co-treatment synergistically increased IL-8 production in comparison to Pam2CSK4 or poly(I:C) alone, implying that co-infection of viruses and bacteria can synergistically induce inflammatory responses in the dental pulp. Taken together, these results suggest that human DPCs potentially sense and respond to viral double-stranded RNAs, leading to effective induction of innate immune responses.

Mechanisms of length-dependent recognition of viral double-stranded RNA by RIG-I

Retinoic acid-inducible gene I (RIG-I) is the most front-line cytoplasmic viral RNA sensor and induces antiviral immune responses. RIG-I recognizes short double-stranded (dsRNA) (< 500 bp), but not long dsRNA (> 500 bp) to trigger antiviral signaling. Since RIG-I is capable of binding with dsRNA irrespective of size, length-dependent RIG-I signaling remains elusive. Here, we demonstrated that RIG-I bound to long dsRNA with slow kinetics. Remarkably, RIG-I/short dsRNA complex efficiently dissociated in an ATP hydrolysis-dependent manner, whereas RIG-I/long dsRNA was stable and did not dissociate. Our study suggests that the dissociation of RIG-I from RIG-I/dsRNA complex could be a step for efficient antiviral signaling. Dissociated RIG-I exhibited homo-oligomerization, acquiring ability to physically associate with MAVS, and biological activity upon introduction into living cells. We herein discuss common and unique mechanisms of viral dsRNA recognition by RIG-I and MDA5.

Infection Defects of RNA and DNA Viruses Induced by Antiviral RNA Interference.

Immune recognition of viral genome-derived double-stranded RNA (dsRNA) molecules and their subsequent processing into small interfering RNAs (siRNAs) in plants, invertebrates, and mammals trigger specific antiviral immunity known as antiviral RNA interference (RNAi). Immune sensing of viral dsRNA is sequence-independent, and most regions of viral RNAs are targeted by virus-derived siRNAs which extensively overlap in sequence. Thus, the high mutation rates of viruses do not drive immune escape from antiviral RNAi, in contrast to other mechanisms involving specific virus recognition by host immune proteins such as antibodies and resistance (R) proteins in mammals and plants, respectively. Instead, viruses actively suppress antiviral RNAi at various key steps with a group of proteins known as viral suppressors of RNAi (VSRs). Some VSRs are so effective in virus counter-defense that potent inhibition of virus infection by antiviral RNAi is undetectable unless the cognate VSR is rendered nonexpressing or nonfunctional. Since viral proteins are often multifunctional, resistance phenotypes of antiviral RNAi are accurately defined by those infection defects of VSR-deletion mutant viruses that are efficiently rescued by host deficiency in antiviral RNAi. Here, we review and discuss in vivo infection defects of VSR-deficient RNA and DNA viruses resulting from the actions of host antiviral RNAi in model systems.

Omicron-induced interferon signaling prevents influenza A H1N1 and H5N1 virus infection.

Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface cultures of primary human bronchial epithelial cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active types I (α/β) and III (λ) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signaling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knockout prevented it. The JAK/STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/STAT signaling. In conclusion, our study (1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, (2) shows that Omicron infection protects cells from influenza A virus super-infection, and (3) indicates that BA.1 and BA.5 induce comparable antiviral states.

RNA interference, an emerging component of antiviral immunity in mammals.

Antiviral RNA interference (RNAi) is an immune pathway that can, in certain conditions, protect mammalian cells against RNA viruses. It depends on the recognition and dicing of viral double-stranded RNA by a protein of the Dicer family, which leads to the production of viral small interfering RNAs (vsiRNAs) that sequence-specifically guide the degradation of cognate viral RNA. If the first line of defence against viruses relies on type-I and type-III interferons (IFN) in mammals, certain cell types such as stem cells, that are hyporesponsive for IFN, instead use antiviral RNAi via the expression of a specific antiviral Dicer. In certain conditions, antiviral RNAi can also contribute to the protection of differentiated cells. Indeed, abundant vsiRNAs are detected in infected cells and efficiently guide the degradation of viral RNA, especially in cells infected with viruses disabled for viral suppressors of RNAi (VSRs), which are virally encoded blockers of antiviral RNAi. The existence and importance of antiviral RNAi in differentiated cells has however been debated in the field, because data document mutual inhibition between IFN and antiviral RNAi. Recent developments include the engineering of a small molecule inhibitor of VSR to probe antiviral RNAi in vivo, as well as the detection of vsiRNAs inside extracellular vesicles in the serum of infected mice. It suggests that using more complex, in vivo models could allow to unravel the contribution of antiviral RNAi to immunity at the host level.

TLR3 Recognition of Viral Double-Stranded RNA in Human Dental Pulp Cells is Important for the Innate Immunity

TLR3 Recognition of Viral Double-Stranded RNA in Human Dental Pulp Cells is Important for the Innate Immunity

Altered RNA Editing in Atopic Dermatitis Highlights the Role of Double-Stranded RNA for Immune Surveillance

Atopic dermatitis (AD) is associated with dysregulated type 1 IFN‒mediated responses, in parallel with the dominant type 2 inflammation. However, the pathophysiology of this dysregulation is largely unknown. Adenosine-to-inosine RNA editing plays a critical role in immune regulation by preventing double-stranded RNA recognition by MDA5 and IFN activation. We studied global adenosine-to-inosine editing in AD to elucidate the role played by altered editing in the pathophysiology of this disease. Analysis of three RNA-sequencing datasets of AD skin samples revealed reduced levels of adenosine-to-inosine RNA editing in AD. This reduction was seen globally throughout Alu repeats as well as in coding genes and in specific pre-mRNA loci expected to create long double-stranded RNA, the main substrate of MDA5 leading to type I IFN activation. Consistently, IFN signature genes were upregulated. In contrast, global editing was not altered in systemic lupus erythematosus and systemic sclerosis, despite IFN activation. Our results indicate that altered editing leading to impairment of the innate immune response may be involved in the pathogenesis of AD. Possibly, it may be relevant for additional autoimmune and inflammatory diseases.

Selected Articles from This Issue

Ewing sarcoma is characterized by oncogenic gene fusions and elevated lysine-specific demethylase-1 (LSD1) expression. While Theisen and colleagues previously devised a small molecule LSD1 inhibitor that demonstrates therapeutic efficacy against Ewing sarcoma — SP-2509 — treatment with SP-2509 alone potentiates drug resistance. In this study, Tokarsky et al., sought to elucidate molecular mechanisms underlying SP-2509 resistance by using a CRISPR–Cas9 loss-of-function screen in SP-2509–sensitive Ewing sarcoma cell lines. Results from the screen demonstrated that abrogating expression of mitochondrial ribosomal protein L45 (MRPL45) as well as members of electron transport chain complexes III (CIII) and IV (CIV) reduces SP-2509 responsiveness. Accordingly, CIII inhibitors and targeted removal of genes encoding CIII constituents ubiquinone-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1) and cytochrome c1 (CYC1) using CRISPR–Cas9 induces SP-2509 resistance in Ewing sarcoma cell lines. Silencing UQCRFS1, CYC1, or MRPL45 expression diminishes transcriptional responses to SP-2509, indicating that mitochondrial function contributes to transcriptional programs underlying SP-2509 cytotoxicity. Overall, this study implicates mitochondrial dysfunction as a mechanism facilitating SP-2509 resistance, providing a target that may augment SP-2509 efficacy.Germline BRCA1/2 mutations increase the risk of multiple cancers that exhibit homologous recombination deficiency (HRD). BRCA1/2 mutations and subsequent HRD are associated with a number of genomic mutational signatures that have been previously described and seem to represent use of backup DNA repair pathways. These signatures, such as HRDetect and others, have been quantified and used to inform prognoses and therapeutic strategies. In this study, Moore and colleagues used current pre-clinical knowledge about polymerase theta-mediated end joining (TMEJ) and the genomic scars most typical of this backup repair pathway to determine if TMEJ signatures have prognostic value in advanced ovarian cancer. The authors found that both a TMEJ deletion signature with 4 or more base pair stretches of microhomology (TMEJ4) and TMEJ-associated templated insertions (TINS) are enriched in tumors with BRCA1/2 mutations. The authors also discovered that patients displaying high HRDetect and TINS signatures survive significantly longer than patients displaying one or neither signature. Taken together, this study highlights the potential utility of TMEJ mutational signatures in advanced ovarian cancer prognosis.Immune checkpoint inhibitors (ICI) are ineffective in pancreatic cancer, rendering combination treatments capable of augmenting ICI imperative. Radiation and inhibition of DNA damage response proteins, such as DNA-dependent protein kinase (DNA-PK), can activate antitumor immunity. However, whether and how DNA-PK inhibition mediates antitumor immunity in typically immunology “cold” cancers, including pancreatic cancer, is unknown. In their study, Wang and colleagues used radiation and DNA-PK inhibitor M3814 in mouse pancreatic cancer models to test whether the combination regimen can enhance antitumor immunity. The authors found that radiation-M3814 treatment induces type I interferon (T1IFN) signaling and slows tumor growth uniquely in mice with intact immune systems. The authors demonstrated that radiation and M3814-mediated T1IFN signaling is dependent on RNA polymerase III (POL III)-retinoic acid-inducible gene I (RIG-I)-mitochondrial anti-viral-signaling protein (MAVS) signaling, presumably through production and recognition of double-stranded RNA. Furthermore, T1IFN signaling induces tumor PD-L1 expression, and radiation-M3814 treatment augments anti-PD-L1 efficacy in vivo. In sum, this study elucidates a novel mechanism by which ICI efficacy may be enhanced in typically immunologically “cold” pancreatic cancers.Increased MYC stabilization is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), but molecular mechanisms underlying MYC stabilization in T-ALL are incompletely understood. To study effects of two MYC mutations — T58A and S62A — on MYC stabilization and MYC-induced pathophysiology in T-ALL, Daniel and colleagues generated T-cell–specific MycWT, MycT58A, and MycS62A knock-in mouse models in which endogenous wild-type Myc can be maintained or eliminated. The authors observed that MycT58A induces heightened ex vivo T-cell proliferation and thymic mass relative to MycS62A, and that MycT58A causes T-cell lymphomagenesis more rapidly than does MycS62A. Nonetheless, MycS62A does induce clonal T-cell lymphomas that resemble T-ALL and, unlike MycT58A, causes non-transgene driven, incidental thymic B-cell lymphomas with splenomegaly. Differential phenotypes engendered by MycT58A and MycS62A are accompanied by disparate transcriptional programs. MycT58A is associated with altered expression of genes underlying angiogenesis and inflammation, and MycS62A displays changes in expression of genes involved in maintaining genomic stability. Altogether, this study demonstrates that MycT58A and MycS62A induce unique pathophysiology in T-cell lymphomas, which may inform future research and T-ALL disease management strategies.

Inhibition of PI3Kδ Differentially Regulates Poly I:C- and Human Metapneumovirus-Induced PD-L1 and PD-L2 Expression in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNβ or IFNλ increased PD-L1 and PD-L2, and IFNβ or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C–induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C–induced PD-L1. IC87114 enhanced poly I:C–induced gene expression of IFNβ, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C–induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C–induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus–infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.

Inhibition of PKR by Viruses.

Cells respond to viral infections through sensors that detect non-self-molecules, and through effectors, which can have direct antiviral activities or adapt cell physiology to limit viral infection and propagation. Eukaryotic translation initiation factor 2 alpha kinase 2, better known as PKR, acts as both a sensor and an effector in the response to viral infections. After sensing double-stranded RNA molecules in infected cells, PKR self-activates and majorly exerts its antiviral function by blocking the translation machinery and inducing apoptosis. The antiviral potency of PKR is emphasized by the number of strategies developed by viruses to antagonize the PKR pathway. In this review, we present an update on the diversity of such strategies, which range from preventing double-stranded RNA recognition upstream from PKR activation, to activating eIF2B downstream from PKR targets.

Mechanisms and applications of peptide nucleic acids selectively binding to double-stranded RNA.

RNAs form secondary structures containing double-stranded base paired regions and single-stranded regions. Probing, detecting and modulating RNA structures and dynamics requires the development of molecular sensors that can differentiate the sequence and structure of RNAs present in viruses and cells, as well as in extracellular space. In this review, we summarize the recent progress on the development of chemically modified peptide nucleic acids (PNAs) for the selective recognition of double-stranded RNA (dsRNA) sequences over both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) sequences. We also briefly discuss the applications of sequence-specific dsRNA-binding PNAs in sensing and stabilizing dsRNA structures and inhibiting dsRNA-protein interactions.

Immune Clearance of Senescent Cells Via Activation of Endogenous Retroviruses and the Viral Recognition Pathway

Expression of genomic repeat element (RE) is known to induce interferon signaling termed ‘viral mimicry’. RE activates double-stranded RNA (dsRNA) recognition via mitochondrial antiviral signaling protein (MAVS), leading to activation of interferon-stimulated genes (ISG) and subsequent cell death. We recently showed that the expression of RE is dysregulated during acute myeloid leukemia (AML) development and in myelodysplastic syndrome (MDS). RE expression is suppressed in leukemic stem cells (LSCs) compared to pre-leukemic stem cells and blasts. Similarly, high-grade MDS showed significant suppression of RE and immune pathways compared to low-grade MDS. These findings suggest that the viral recognition pathway plays a significant role in hematopoietic disorders.Senescent cells activate interferon response and secrete inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). To investigate whether the inflammatory phenotype seen in senescent cells is mediated by RE expression,we analyzed the RNA sequencing data of senescent cell lines for expression of RE. We found significant upregulation of REs following multiple modalities of senescence induction. We induced senescence in Human HSPCs and 3 different cell lines (K562, HCT116 and IMR90) using an alkylating agent, mitomycin (MMC), and/or radiation, and found significant upregulation of the measured REs. dsRNA is known to trigger the type 1 interferon response through the dsRNA sensor RIG-1/MDA5. Upon recognition of dsRNA by RIG-1, MDA5 is activated, which in turn leads to aggregation of MAVS in the outer mitochondrial membrane. Following senescence, we found aggregation of MAVS in the mitochondrial fraction of murine HSPCs and HCT116 cell. Aggregated MAVS phosphorylates IRF3 and IRF7, leading to translocation of IRF3, IRF7, and NF-κB into the nucleus, where these proteins induce expression of ISGs. Western blots of MMC-treated murine HSPCs showed increase in nuclear NF-κB and p-IRF7. qRT-PCR analysis of murine HSPCs and HCT116, IMR90, and K562 cells following MMC treatment showed increased expression of ISGs and NK ligands, suggesting activation of the dsRNA recognition pathway along with the transcription of ISGs and NK ligands upon senescence induction.We examined whether senescent cells activate NK cells. Mononuclear cells were isolated from the blood of healthy donors when incubated with HCT116 and IMR90 cells following MMC treatment, showed activation of NK cells (6-10-fold). To study NK-mediated cell killing of senescent cells, we incubated senescent IMR90 cells (target cells) with NK92Mi (human NK) cells. MMC-treated IMR90 cells were reduced by 40%. No significant reduction in mock cells was observed. This suggests that activation of ds-RNA recognition pathway in senescence (like in viral infection) leads to up-regulation of NK ligands, NK cell activation, and clearance of senescent cells by NK cells. In order to investigate if MAVS is critical for senescence mediated immune clearance, we employed cell lines with knock out or knock down of MAVS. Abrogation of the dsRNA recognition pathway diminished the interferon response, NK ligand expression and NK mediated clearance of senescent cells.Finally, to evaluate if senescence in human HSPCs also increases RE expression in vivo, we isolated human senescent HSPCs from healthy donors' blood using senescence associated □-galactosidase (SA-β-gal) and did RNA sequencing. Human senescent HSPCs showed overexpression of RE along with pathways of inflammation. % of SA-β-gal+ cells in HSPCs from 26 donors between 20 and 71 years of age (median = 39 years) showed no significant correlation with age (P= 0.25, R2 = 0.06), suggesting that senescent HSPCs do not accumulate with age; implying they are cleared as they are formed. Taken together, these findings suggest that RE expression and the subsequent activation of the dsRNA recognition pathway plays an important role in maintaining homeostasis during aging and dysregulation of this pathway could play a role in hematopoietic disorders such as AML and MDS. DisclosuresNo relevant conflicts of interest to declare.

Pyridazine Nucleobase in Triplex-Forming PNA Improves Recognition of Cytosine Interruptions of Polypurine Tracts in RNA.

Sequence specific recognition of regulatory noncoding RNAs would open new possibilities for fundamental science and medicine. However, molecular recognition of such complex double-stranded RNA (dsRNA) structures remains a formidable problem. Recently, we discovered that peptide nucleic acids (PNAs) form an unusually stable and sequence-specific triple helix with dsRNA. Triplex-forming PNAs could become universal tools for recognition of noncoding dsRNAs but are limited by the requirement of polypurine tracts in target RNAs as only purines form stable Hoogsteen hydrogen bonded base triplets. Herein, we systematically surveyed simple nitrogen heterocycles PN as modified nucleobases for recognition of cytosine in PN*C-G triplets. We found that a 3-pyridazinyl nucleobase formed significantly more stable PN*C-G triplets than other heterocycles including the pyrimidin-2-one previously used by us and others for recognition of cytosine interruptions in polypurine tracts of PNA-dsRNA triplexes. Our results improve triple helical recognition of dsRNA and provide insights for future development of new nucleobases to expand the sequence scope of noncoding dsRNAs that can be targeted by triplex-forming PNAs.

TDP-43 prevents endogenous RNAs from triggering a lethal RIG-I-dependent interferon response.

SUMMARYRIG-I-like receptors (RLRs) are involved in the discrimination of self versus non-self via the recognition of double-stranded RNA (dsRNA). Emerging evidence suggests that immunostimulatory dsRNAs are ubiquitously expressed but are disrupted or sequestered by cellular RNA binding proteins (RBPs). TDP-43 is an RBP associated with multiple neurological disorders and is essential for cell viability. Here, we demonstrate that TDP-43 regulates the accumulation of immunostimulatory dsRNA. The immunostimulatory RNA is identified as RNA polymerase III transcripts, including 7SL and Alu retrotransposons, and we demonstrate that the RNA-binding activity of TDP-43 is required to prevent immune stimulation. The dsRNAs activate a RIG-I-dependent interferon (IFN) response, which promotes necroptosis. Genetic inactivation of the RLR-pathway rescues the interferon-mediated cell death associated with loss of TDP-43. Collectively, our study describes a role for TDP-43 in preventing the accumulation of endogenous immunostimulatory dsRNAs and uncovers an intricate relationship between the control of cellular gene expression and IFN-mediated cell death.