Cellular origins of dsRNA, their recognition and consequences

Abstract

Double-stranded RNA (dsRNA) is associated with most viral infections - it either constitutes the viral genome (in the case of dsRNA viruses) or is generated in host cells during viral replication. Hence, nearly all organisms have the capability of recognizing dsRNA and mounting a response, the primary aim of which is to mitigate the potential infection. In vertebrates, a set of innate immune receptors for dsRNA induce a multitude of cell-intrinsic and cell-extrinsic immune responses upon dsRNA recognition. Notably, recent studies showed that vertebrate cells can accumulate self-derived dsRNAs or dsRNA-like species upon dysregulation of several cellular processes, activating the very same immune pathways as in infected cells. On the one hand, such aberrant immune activation in the absence of infection can lead to pathogenesis of immune disorders, such as Aicardi-Goutières syndrome. On the other hand, the same innate immune reaction can be induced in a controlled setting for a therapeutic benefit, as occurs in immunotherapies. In this Review, we describe mechanisms by which immunostimulatory dsRNAs are generated in mammalian cells, either by viruses or by the host cells, and how cells respond to them, with the focus on recent developments regarding the role of cellular dsRNAs in immune modulation.

Fig. 1 |. dsRNA sensors and their signalling.

Double stranded RNAs (dsRNAs) from either viral or cellular origins induce three types of cellular responses. They are not mutually exclusive and may occur within the same cell. a | The first type of response is classical antiviral innate immune responses mediated by RIG-I or MDA5 (RIG-I-like receptors (RLRs)) and Toll-like receptor 3 (TLR3). RLRs in the cytoplasm and TLRs in the endosome detect dsRNAs and form signalling-competent oligomers to activate their respective downstream adaptors, mitochondrial antiviral-signalling protein (MAVS) and TRIF. Upon dsRNA binding, RLRs form filaments along the length of dsRNA, which promotes oligomerization of their caspase activation and recruitment domains (CARDs) and triggers MAVS filament formation for downstream signal activation. RIG-I may also be stimulated by RNAs besides long dsRNA by forming non-filamentous multimers, but the structural features of such RNAs and RIG-I multimers are unclear. These pathways then converge by activating the common downstream signalling molecules, such as TNF receptor-associated factors (TRAFs) and TANK-binding kinase 1 (TBK1), culminating in the activation of the transcription factors interferon-regulatory factor 3 (IRF3) and NF-κB for producing type I interferons and other proinflammatory cytokines. b | The second type of response induces global inhibition of protein synthesis and thus cell growth. This response is mediated by protein kinase R (PKR) and oligoadenylate synthases (OASes), which become active upon binding to dsRNA. PKR activation via dimerization and autophosphorylation results in phosphorylation of a key translation initiation factor (eIF2α) and subsequent inhibition of most protein synthesis. Activated OASes synthesize 2′−5′-linked oligoadenylate (2–5An), which serves as a soluble second messenger to activate ribonuclease L (RNase L). RNase L in turn degrades the bulk of cytosolic RNAs, including mRNA, ribosomal RNA (rRNA) and tRNA, resulting in translation inhibition. c | The third type of response to dsRNA is mediated by the NOD-, LRR- and pyrin domain-containing 1 (NLRP1) inflammasome, a macromolecular complex containing the receptor NLRP1, the adaptor ASC and the effector caspase 1. Upon dsRNA binding, NLRP1 triggers release of its UPA and CARD domains, which then assembles the inflammasome seed, inducing inflammasome formation and activating caspase 1. Activated caspase 1 then cleaves precursors of inflammatory cytokines (such as IL-1β and IL-18) and a pore-forming protein gasdermin D (GSDMD). The GSDMD pore forms in the plasma membrane and induces pyroptosis, the inflammatory form of cell death.

Fig. 2 |. RNA modifications affect the RNA’s secondary structures and interaction with immune sensors.

The canonical nucleosides adenosine, uridine and cytidine can be modified by enzymes that install new chemical groups (shown in red). The RNA modifications can change base-pairing interactions, protein binding and secondary structures, which can prevent the modified RNAs from forming immunogenic structures, such as double-stranded RNAs (dsRNAs), and evading detection by immune sensors, including Toll-like receptors (TLRs), RIG-I and protein kinase R (PKR). ADAR, double-stranded RNA-specific adenosine deaminase.

Fig. 3 |. Endogenous sources of dsRNA and cellular regulatory processes.

Cells have diverse endogenous sources of double-stranded RNA (dsRNA) and utilize multiple mechanisms to suppress its biogenesis and accumulation. a | Epigenetic derepression of transposable elements (TEs), such as endogenous retroviruses (ERVs), long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), can lead to dsRNA generation. These elements can be transcribed in a bidirectional manner or as an inverted repeat, forming sense–antisense hybrid or fold-back hairpin dsRNA. Biogenesis of TE-based dsRNAs is normally suppressed by epigenetic silencing mechanisms involving DNA methyltransferases (DNMTs), the histone H3 K9 methyltransferase SETDB1, its partner the human silencing hub (HUSH) and the histone demethylase LSD1. The only exception is inverted repeat Alus (IR-Alus), some of which are constitutively produced within the 3′ untranslated region of many mRNAs. b | Once transcribed, cellular RNAs are regulated by post-transcriptional modifications, such as adenosine deamination to inosine (Ino) and N⁶-adenosine methylation (to produce N⁶-methyladenosine (m⁶A)), both of which disrupt the structure of dsRNA and lower its immunogenicity (shown by inhibitory arrows). Deregulation of these (and potentially other) RNA modifications can result in the recognition of normal cellular transcripts as foreign, owing to the formation of local duplex structures. Splicing inhibition can lead to an increase in the levels of dsRNAs, as a result of the increase in the levels of transcripts with retained introns, which may form double-stranded structures. Splicing is also associated with the generation of circular RNAs (circRNAs), which can form dsRNA structures more easily than their linear counterparts. On the one hand, these dsRNA structures can be recognized by RIG-I, but this is negatively regulated by m⁶A modification, normally present in circRNAs. On the other hand, circRNAs can also act as protein sponges and sequester protein kinase R (PKR) and prevent its activation in sterile conditions. c | RNA degradation mechanisms, such as those involving Dicer, RNA exosome complex and the lysosomal RNA transporter SIDT2, may prevent excessive accumulation of dsRNA through poorly understood mechanisms (question marks). d | Genotoxic stress (for example, resulting from exposure to ionizing radiation) and aberrant activation of RNA polymerase III (Pol III; for example when MYC is activated in cancer) can promote biogenesis of RLR-stimulatory dsRNAs, but the precise nature of these dsRNAs remains unclear (question marks). These may be aberrantly processed RNA components of the spliceosome (U1 and U2 small nuclear RNAs) or products of Pol III which contain 5′-triphosphate (5′ppp). The recognition of the latter could be regulated by the phosphatase DUSP11, which can remove 5′ppp. e | Mitochondria are a rich source of dsRNA as mitochondrial RNAs are produced by bidirectional transcription of the circular DNA. Normally, the level of mitochondrial dsRNA is regulated by the mitochondrial RNA degradosome, which includes the nuclease polynucleotide phosphorylase (PNPase) and the helicase SUV3. During mitochondrial dysregulation, mitochondrial dsRNA can gain access to cytosolic dsRNA sensors and activate them through a poorly understood mechanism. OAS, oligoadenylate synthase; TLR3, Toll-like receptor 3.

Fig. 4 |. Consequences of dsRNA recognition.

Sterile activation of double-stranded RNA (dsRNA) sensors can occur in normal, pathologic and therapeutic conditions. Here, we use an immunological threshold model to summarize examples in each category. dsRNA sensors have an evolutionarily optimized activation threshold that allows the receptors to tolerate a certain level and certain kinds of dsRNAs (for example, dsRNA with short and imperfect complementarity is normally tolerated by MDA5), while those RNAs beyond the threshold (for example, viral dsRNA) would activate the dsRNA sensors. In normal conditions, the levels of cellular dsRNAs are well below the activation threshold of dsRNA sensors. However, there are cases where a subset of cellular dsRNAs breach the threshold in a transient and controlled fashion, and innate immune functions of the dsRNA sensors are integrated into the normal biological processes. This includes protein kinase R (PKR) activation during mitosis, neuronal excitation in the brain and Toll-like receptor 3 (TLR3) activation during tissue regeneration in the skin. By contrast, constitutive and uncontrolled breaching of the tolerance threshold leads to pathogenesis of immune disorders and other diseases. This can occur through gain-of-function (GOF) mutations of the dsRNA sensor, which lowers the threshold, leading to misrecognition of otherwise inert cellular dsRNA. Such cases include Aicardi–Goutières syndrome (AGS), Singleton–Merten syndrome (SMS) and systemic lupus erythematosus (SLE) caused by GOF RIG-I-like receptors (RLRs) and dystonia caused by GOF PKR. Alternatively, similar diseases can be caused by loss-of-function (LOF) mutations in the regulators. For example, LOF in ADAR1 causes AGS through constitutive activation of RLRs, PKR or oligoadenylate synthases (OASes), LOF in SKIV2 causes trichohepatoenteric syndrome (THES), where constitutive activation of RIG-I is thought to contribute, LOF in PACT causes dystonia through constitutive activation of PKR and LOF in Dicer has been associated with age-related macular degeneration (AMD), although in the last case the exact sensor involved has not been determined. In addition, increased generation of immunostimulatory dsRNAs has been observed in cancer, where aberrant activation of RNA polymerase III (Pol III) downstream of the activity of the oncogenic protein MYC leads to increased generation of RNAs prone to forming secondary structures and RNAs containing 5′-triphosphate, which are recognized by RIG-I. Finally, immune functions of dsRNA sensors can be leveraged in cancer immunotherapy. Chemotherapy and radiotherapy were shown to confer anticancer efficacy partly by inducing biogenesis of immunostimulatory dsRNAs, which activate a broad range of dsRNA sensors.