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RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs) 1 , 2 . However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3′ overhang and a terminal loop 3 – 11 . Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis -acting element, termed the ‘GYM motif’ (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified ‘ruler’-like counting mechanisms from the 5′ and 3′ ends of pre-miRNA 3 – 6 . Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics. Massively parallel assays reveal a highly conserved sequence motif termed the GYM motif, which potentiates RNA interference by directing Dicer-mediated small RNA processing.

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites 1 – 4 . However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis , we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming. Double-stranded RNA structures downstream of start codons play a role in translation initiation by regulating start-codon selection in plant immune responses, and also contribute to translational reprogramming in mammalian systems.

Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs) 1 – 4 . These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR 5 . Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3′ untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers. A small molecule can bypass the RNA-editing enzyme ADAR1 to directly activate the Z-form nucleic acid sensor ZBP1, induce necroptosis in tumour fibroblasts and reverse resistance to immune checkpoint blockade in mouse models of melanoma.

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor 1 – 6 . This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses 7 , 8 . However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of ‘orphan’ CpG islands 9 . In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA 10 , which prevents activation of the MDA5 receptor 11 . We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment. Inverted-repeat Alu elements are the main source of drug-induced immunogenic double-stranded RNAs, which are destabilized by the RNA deaminase ADAR1, thereby limiting activation of the immune response.

In mammals, cyclic GMP–AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2′3′-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes 1 and eukaryotes 2 – 5 . In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections 6 – 8 . Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster . We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3′2′-cGAMP, whereas cGLR2 produces a combination of 2′3′-cGAMP and 3′2′-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2′3′-cGAMP. Two cGAS-like receptors, cGLR1 and cGLR2, identified in Drosophila melanogaster are shown to induce antiviral immunity in response to RNA or DNA virus infections through the production of 2′3′-cGAMP and 3′2′-cGAMP.

In the absence of DHX9, circular RNAs accumulate and transcription and translation are dysregulated—effects that are exacerbated by concomitant depletion of the RNA-editing enzyme ADAR. DHX9 suppresses Alu-derived defects In the human genome, there are more than a million copies of the Alu transposable element. Movement of Alu elements is a common source of mutations, but as insertions usually occur in non-coding regions, they are often without discernible effect. Alu elements located near one another in an inverted orientation will form secondary structures that may affect various nuclear processes. Asifa Akhtar and colleagues find that the RNA helicase, DHX9, binds transcribed ‘IRAlus’ (inverted repeat Alu elements). In the absence of DHX9, circular RNAs accumulate, and transcription and translation are dysregulated. These effects are further exacerbated by co-depletion of DHX9 and ADAR p150, an interferon-inducible RNA modification enzyme. The authors conclude that these proteins protect against transposon insertion, which can have deleterious effects on gene expression. Transposable elements are viewed as ‘selfish genetic elements’, yet they contribute to gene regulation and genome evolution in diverse ways 1 . More than half of the human genome consists of transposable elements 2 . Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome 2 . Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability 3 . Alu elements are the main targets of the RNA-editing enzyme ADAR 4 and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC 5 , but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant 6 nuclear RNA helicase 7 , binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

IgA secretion at mucosal sites is important for host defence against pathogens as well as maintaining the symbiosis with microorganisms present in the small intestine that affect IgA production. In the present study, we tested the ability of 5 strains of lactic acid bacteria stimulating IgA production, being Pediococcus acidilactici K15 selected as the most effective on inducing this protective immunoglobulin. We found that this response was mainly induced via IL-10, as efficiently as IL-6, secreted by K15-stimulated dendritic cells. Furthermore, bacterial RNA was largely responsible for the induction of these cytokines; double-stranded RNA was a major causative molecule for IL-6 production whereas single-stranded RNA was critical factor for IL-10 production. In a randomized, double-blind, placebo-controlled clinical trial, ingestion of K15 significantly increased the secretory IgA (sIgA) concentration in saliva compared with the basal level observed before this intervention. These results indicate that functional lactic acid bacteria induce IL-6 and IL-10 production by dendritic cells, which contribute to upregulating the sIgA concentration at mucosal sites in humans.

Plant-mediated RNA interference (RNAi) shows great potential in crop protection. It relies on plants stably expressing double-stranded RNAs (dsRNAs) that target essential genes in pest insects. Practical application of this strategy is challenging because producing sufficient amounts of stable dsRNA in plants has proven to be difficult to achieve with conventional transgenesis. In addition, many insects do not respond to exogenously applied dsRNAs, either degrading them or failing to import them into the cytoplasm. We summarize recent progress in RNAi-mediated insect pest control and discuss factors determining its efficacy. Expressing dsRNA in chloroplasts overcomes many of the difficulties previously encountered. We also highlight remaining challenges and discuss the environmental and biosafety issues involved in the use of this technology in agriculture. Plant-mediated RNAi that targets essential genes in insects and other pests is becoming a promising approach in crop protection. Expression of dsRNA targeting insect genes can potentially provide crop protection without chemical pesticides and offers the additional advantages that no foreign protein is made and the number of target genes is nearly unlimited. The length and amount of the dsRNA as well as its stability in planta and in the gut of the target insect are crucial factors determining the success of plant-mediated RNAi strategies. High-level expression of long dsRNAs from the genome of the chloroplast represents a particularly promising strategy for efficient RNAi-mediated crop protection.

In the past two decades, emerging studies have suggested that DExD/H box helicases belonging to helicase superfamily 2 (SF2) play essential roles in antiviral innate immunity. However, the antiviral functions of helicase SF1, which shares a conserved helicase core with SF2, are little understood. Here we demonstrate that zinc finger NFX1-type containing 1 (ZNFX1), a helicase SF1, is an interferon (IFN)-stimulated, mitochondrial-localised dsRNA sensor that specifically restricts the replication of RNA viruses. Upon virus infection, ZNFX1 immediately recognizes viral RNA through its Armadillo-type fold and P-loop domain and then interacts with mitochondrial antiviral signalling protein to initiate the type I IFN response without depending on retinoic acid-inducible gene I-like receptors (RLRs). In short, as is the case with interferon-stimulated genes (ISGs) alone, ZNFX1 can induce IFN and ISG expression at an early stage of RNA virus infection to form a positively regulated loop of the well-known RLR signalling. This provides another layer of understanding of the complexity of antiviral immunity. Wang et al. identify ZNFX1 as a mitochondria-localised sensor that recognizes viral dsRNA and induces a type I interferon response, thereby restricting virus infection.

In flies, Argonaute2 (Ago2) and small interfering RNA (siRNA) form an RNA-induced silencing complex to repress viral transcripts 1 . The RNase III enzyme Dicer-2 associates with its partner protein R2D2 and cleaves long double-stranded RNAs to produce 21-nucleotide siRNA duplexes, which are then loaded into Ago2 in a defined orientation 2 – 5 . Here we report cryo-electron microscopy structures of the Dicer-2–R2D2 and Dicer-2–R2D2–siRNA complexes. R2D2 interacts with the helicase domain and the central linker of Dicer-2 to inhibit the promiscuous processing of microRNA precursors by Dicer-2. Notably, our structure represents the strand-selection state in the siRNA-loading process, and reveals that R2D2 asymmetrically recognizes the end of the siRNA duplex with the higher base-pairing stability, and the other end is exposed to the solvent and is accessible by Ago2. Our findings explain how R2D2 senses the thermodynamic asymmetry of the siRNA and facilitates the siRNA loading into Ago2 in a defined orientation, thereby determining which strand of the siRNA duplex is used by Ago2 as the guide strand for target silencing. Cryo-electron microscopy structures of Drosophila Dicer-2–R2D2 complexes with and without small interfering RNA reveal how the RNA is presented to Argonaute in the correct orientation for viral gene silencing.