Structure and Degradation of Circular RNAs Regulate PKR Activation in Innate Immunity.

Circular RNAs (circRNAs) produced from back-splicing of exons of pre-mRNAs are widely expressed, but current understanding of their functions is limited. These RNAs are stable in general and are thought to have unique structural conformations distinct from their linear RNA cognates. Here, we show that endogenous circRNAs tend to form 16-26 bp imperfect RNA duplexes and act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity. Upon poly(I:C) stimulation or viral infection, circRNAs are globally degraded by RNase L, a process required for PKR activation in early cellular innate immune responses. Augmented PKR phosphorylation and circRNA reduction are found in peripheral blood mononuclear cells (PBMCs) derived from patients with autoimmune disease systemic lupus erythematosus (SLE). Importantly, overexpression of the dsRNA-containing circRNA in PBMCs or T cells derived from SLE can alleviate the aberrant PKR activation cascade, thus providing a connection between circRNAs and SLE.

Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown.

Type I interferon (IFN) is produced when host sensors detect foreign nucleic acids, but how sensors differentiate self from nonself nucleic acids, such as double-stranded RNA (dsRNA), is incompletely understood. Mutations in ADAR1, an adenosine-to-inosine editing enzyme of dsRNA, cause Aicardi-Goutières syndrome, an autoinflammatory disorder associated with spontaneous interferon production and neurologic sequelae. We generated ADAR1 knockout human cells to explore ADAR1 substrates and function. ADAR1 primarily edited Alu elements in RNA polymerase II (pol II)-transcribed mRNAs, but not putative pol III-transcribed Alus. During the IFN response, ADAR1 blocked translational shutdown by inhibiting hyperactivation of PKR, a dsRNA sensor. ADAR1 dsRNA binding and catalytic activities were required to fully prevent endogenous RNA from activating PKR. Remarkably, ADAR1 knockout neuronal progenitor cells exhibited MDA5 (dsRNA sensor)-dependent spontaneous interferon production, PKR activation, and cell death. Thus, human ADAR1 regulates sensing of self versus nonself RNA, allowing pathogen detection while avoiding autoinflammation.

The competitive landscape of the dsRNA world.

The ability to sense and respond to infection is essential for life. Viral infection produces double-stranded RNAs (dsRNAs) that are sensed by proteins that recognize the structure of dsRNA. This structure-based recognition of viral dsRNA allows dsRNA sensors to recognize infection by many viruses, but it comes at a cost-the dsRNA sensors cannot always distinguish between "self" and "nonself" dsRNAs. "Self" RNAs often contain dsRNA regions, and not surprisingly, mechanisms have evolved to prevent aberrant activation of dsRNA sensors by "self" RNA. Here, we review current knowledge about the life of endogenous dsRNAs in mammals-the biosynthesis and processing of dsRNAs, the proteins they encounter, and their ultimate degradation. We highlight mechanisms that evolved to prevent aberrant dsRNA sensor activation and the importance of competition in the regulation of dsRNA sensors and other dsRNA-binding proteins.

ADAR1p150 prevents MDA5 and PKR activation via distinct mechanisms to avert fatal autoinflammation.

Effective immunity requires the innate immune system to distinguish foreign nucleic acids from cellular ones. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA-editing enzyme ADAR1 to evade being recognized as viral dsRNA by cytoplasmic dsRNA sensors, including MDA5 and PKR. The loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. Additional RNA-editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, whereas loss of the cytoplasmic ADAR1p150 isoform or its dsRNA-binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150-/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5 or PKR alone. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1.

The RNA deaminase ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. Mutations in ADAR, the gene that encodes ADAR1, cause human immune diseases, including Aicardi-Goutières syndrome (AGS). However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here we generated mice with a single amino acid change in ADAR1 that models the most common human ADAR AGS mutation. These Adar mutant mice developed lethal disease that required MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. A small-molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevented immunopathology and rescued the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

Structural and functional basis of mammalian microRNA biogenesis by Dicer.

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.

RNA circles with minimized immunogenicity as potent PKR inhibitors.

Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.

LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response.

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

RNase L Reprograms Translation by Widespread mRNA Turnover Escaped by Antiviral mRNAs.

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-ß. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.

Multifaceted roles of RNA editing enzyme ADAR1 in innate immunity.

Innate immunity must be tightly regulated to enable sensitive pathogen detection while averting autoimmunity triggered by pathogen-like host molecules. A hallmark of viral infection, double-stranded RNAs (dsRNAs) are also abundantly encoded in mammalian genomes, necessitating surveillance mechanisms to distinguish "self" from "nonself." ADAR1, an RNA editing enzyme, has emerged as an essential safeguard against dsRNA-induced autoimmunity. By converting adenosines to inosines (A-to-I) in long dsRNAs, ADAR1 covalently marks endogenous dsRNAs, thereby blocking the activation of the cytoplasmic dsRNA sensor MDA5. Moreover, beyond its editing function, ADAR1 binding to dsRNA impedes the activation of innate immune sensors PKR and ZBP1. Recent landmark studies underscore the utility of silencing ADAR1 for cancer immunotherapy, by exploiting the ADAR1-dependence developed by certain tumors to unleash an antitumor immune response. In this perspective, we summarize the genetic and mechanistic evidence for ADAR1's multipronged role in suppressing dsRNA-mediated autoimmunity and explore the evolving roles of ADAR1 as an immuno-oncology target.

Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA.

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.

PKR Senses Nuclear and Mitochondrial Signals by Interacting with Endogenous Double-Stranded RNAs.

Protein kinase RNA-activated (PKR) induces immune response by sensing viral double-stranded RNAs (dsRNAs). However, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. Here, we capture these dsRNAs by formaldehyde-mediated crosslinking and immunoprecipitation sequencing and find that various noncoding RNAs interact with PKR. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). MtRNAs can form intermolecular dsRNAs owing to bidirectional transcription of the mitochondrial genome and regulate PKR and eIF2α phosphorylation to control cell signaling and translation. Moreover, PKR activation by mtRNAs is counteracted by PKR phosphatases, disruption of which causes apoptosis from PKR overactivation even in uninfected cells. Our work unveils dynamic regulation of PKR even without infection and establishes PKR as a sensor for nuclear and mitochondrial signaling cues in regulating cellular metabolism.

Activation of protein kinase receptor (PKR) plays a pro-viral role in mammarenavirus-infected cells.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.

dsRNA-induced condensation of antiviral proteins modulates PKR activity.

Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.

Viral evasion of PKR restriction by reprogramming cellular stress granules.

Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), an economically devastating pathogen to the world swine industry. Here, we report that this event is to suppress cellular inflammation and is mediated by the viral replicase protein nsp1ß. We show that nsp1ß is a stress-responsive protein, enters virus-induced stress granules (SGs) during infection, and repurposes SGs into a proviral platform, where it co-opts the SG core component G3BP1 to interact with PKR in a regulated manner. RNA interference silencing of G3BP1 or mutation of specific nsp1ß residues (VS19GG) can abolish the antagonization of PKR activation. The viral mutant carrying the corresponding mutations induces elevated level of PKR phosphorylation and pronounced production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-6, and IL-8), whereas small-interfering RNA knockdown of PKR or treatment with C16, a PKR inhibitor, blocks this effect. Thus, PRRSV has evolved a unique strategy to evade PKR restriction to suppress host inflammatory responses.

Maladaptation after a virus host switch leads to increased activation of the pro-inflammatory NF-κB pathway.

Myxoma virus (MYXV) causes localized cutaneous fibromas in its natural hosts, tapeti and brush rabbits; however, in the European rabbit, MYXV causes the lethal disease myxomatosis. Currently, the molecular mechanisms underlying this increased virulence after cross-species transmission are poorly understood. In this study, we investigated the interaction between MYXV M156 and the host protein kinase R (PKR) to determine their crosstalk with the proinflammatory nuclear factor kappa B (NF-κB) pathway. Our results demonstrated that MYXV M156 inhibits brush rabbit PKR (bPKR) more strongly than European rabbit PKR (ePKR). This moderate ePKR inhibition could be improved by hyperactive M156 mutants. We hypothesized that the moderate inhibition of ePKR by M156 might incompletely suppress the signal transduction pathways modulated by PKR, such as the NF-κB pathway. Therefore, we analyzed NF-κB pathway activation with a luciferase-based promoter assay. The moderate inhibition of ePKR resulted in significantly higher NF-κB­dependent reporter activity than complete inhibition of bPKR. We also found a stronger induction of the NF-κB target genes TNFα and IL-6 in ePKR-expressing cells than in bPKR-expressing cells in response to M156 in both transfection and infections assays. Furthermore, a hyperactive M156 mutant did not cause ePKR-dependent NF-κB activation. These observations indicate that M156 is maladapted for ePKR inhibition, only incompletely blocking translation in these hosts, resulting in preferential depletion of short­half-life proteins, such as the NF-κB inhibitor IκBα. We speculate that this functional activation of NF-κB induced by the intermediate inhibition of ePKR by M156 may contribute to the increased virulence of MYXV in European rabbits.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways­interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)­activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung­derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Activation of amygdala prokineticin receptor 2 neurons drives the anorexigenic activity of the neuropeptide PK2.

Energy homeostasis is a complex system involving multiple hormones, neuropeptides, and receptors. Prokineticins (PK1 and PK2) are agonists to two G protein-coupled receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2), which decrease food intake when injected in rodents. The relative contribution of PKR1 and PKR2 to the anorexigenic effect of PK2 and their site of action in the brain have not yet been elucidated. While PKR1 and PKR2 are both expressed in the hypothalamus, a central region involved in the control of energy homeostasis, PKR2 is also present in the amygdala, which has recently been shown to regulate food intake in response to several anorexigenic signals. PKR trafficking and signaling are inhibited by the melanocortin receptor accessory protein 2 (MRAP2), thus suggesting that MRAP2 has the potential to alter the anorexigenic activity of PK2 in vivo. In this study, we investigated the importance of PKR1 and PKR2 for PK2-mediated inhibition of food intake, the brain region involved in this function, and the effect of MRAP2 on PK2 action in vivo. Using targeted silencing of PKR2 and chemogenetic manipulation of PKR2 neurons, we show that the anorexigenic effect of PK2 is mediated by PKR2 in the amygdala and that altering MRAP2 expression in PKR2 neurons modulates the activity of PK2. Collectively, our results provide evidence that inhibition of food intake by PKs is not mediated through activation of hypothalamic neurons but rather amygdala PKR2 neurons and further establishes the importance of MRAP2 in the regulation of energy homeostasis.

Protection of eIF2B from inhibitory phosphorylated eIF2: A viral strategy to maintain mRNA translation during the PKR-triggered integrated stress response.

The integrated stress response (ISR) protects cells from a variety of insults. Once elicited (e.g., by virus infections), it eventually leads to the block of mRNA translation. Central to the ISR are the interactions between translation initiation factors eIF2 and eIF2B. Under normal conditions, eIF2 drives the initiation of protein synthesis through hydrolysis of GTP, which becomes replenished by the guanine nucleotide exchange factor eIF2B. The antiviral branch of the ISR is activated by the RNA-activated kinase PKR which phosphorylates eIF2, thereby converting it into an eIF2B inhibitor. Here, we describe the recently solved structures of eIF2B in complex with eIF2 and a novel escape strategy used by viruses. While unphosphorylated eIF2 interacts with eIF2B in its "productive" conformation, phosphorylated eIF2 [eIF2(αP)] engages a different binding cavity on eIF2B and forces it into the "nonproductive" conformation that prohibits guanine nucleotide exchange factor activity. It is well established that viruses express so-called PKR antagonists that interfere with double-strand RNA, PKR itself, or eIF2. However recently, three taxonomically unrelated viruses were reported to encode antagonists targeting eIF2B instead. For one antagonist, the S segment nonstructural protein of Sandfly fever Sicilian virus, atomic structures showed that it occupies the eIF2(αP)-binding cavity on eIF2B without imposing a switch to the nonproductive conformation. S segment nonstructural protein thus antagonizes the activity of PKR by protecting eIF2B from inhibition by eIF2(αP). As the ISR and specifically eIF2B are central to neuroprotection and a wide range of genetic and age-related diseases, these developments may open new possibilities for treatments.