dsRNA-binding Protein Research Articles

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

Dimerization of ADARs Expands The Range of Substrates That Can Undergo A‐to‐I RNA Editing

RNA editing alters genomically encoded information to generate the transcriptomic diversity required for normal development and proper neuronal function. The adenosine deaminase that act on RNA (ADAR) family of enzymes catalyze adenosine (A)‐to‐inosine (I) RNA editing at millions of sites in the human transcriptome. However, the molecular mechanisms that regulate editing specificity and efficiency are not understood. Using recombinant proteins, we reveal, that unlike all other A‐to‐I editing enzymes, Caenorhabditis elegans ADR‐2 has a low affinity for double‐stranded RNAs (dsRNAs). Using both in vivo and in vitro approaches, our data indicates that ADR‐2 can physically interact with the dsRNA binding protein, ADR‐1, which is also a deaminase‐deficient member of the ADAR family. Our data from RNA‐immunoprecipitation studies of ADR‐2 indicate that ADR‐1 is required for ADR‐2 to stably interact with some target mRNAs in vivo. Furthermore, biochemical editing assays from lysates expressing ADR‐1 mutant proteins indicate that both RNA binding by ADR‐1 and the protein‐protein interaction between ADR‐1 and ADR‐2 promotes RNA editing efficiency. In sum, we have identified a molecular mechanism for regulating the ability of ADAR to both bind target mRNAs and edit. As human ADARs are known to have some overlapping substrate specificities, these results raise the possibility that dimerization between ADAR family members, and perhaps protein‐protein interactions of ADARs with other dsRNA binding proteins, provides the A‐to‐I editing enzymes with a molecular mechanism to specifically recognize editing targets over the thousands of RNAs present in a cell.Support or Funding InformationAmerican Cancer Society Research Scholar Grant (15‐051‐RMC) to H.A.H. Indiana University School of Medicine Start‐up Funds to H.A.H.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loquacious-PD removes phosphate inhibition of Dicer-2 processing of hairpin RNAs into siRNAs

Drosophila Dicer-2 processes RNA substrates into short interfering RNAs (siRNAs). Loquacious-PD (Loqs-PD), a dsRNA-binding protein that associates with Dicer-2, is required for processing of a subset of RNA substrates including hairpin RNAs into siRNAs. Inorganic phosphate—a small molecule present in all cell types—inhibits Dicer-2 from processing precursor of microRNAs (pre-miRNAs), which are processed by Dicer-1. Whether or how Loqs-PD modulates the inhibitory effect of inorganic phosphate on Dicer-2 processing of RNA substrates is unknown. To address this question, I performed in vitro hairpin RNA processing assay with Dicer-2 in the presence or absence of Loqs-PD and/or inorganic phosphate. I found that inorganic phosphate inhibits Dicer-2 alone, but not Dicer-2 + Loqs-PD, from processing blunt-end hairpin RNAs into siRNAs. Thus, Loqs-PD removes the inhibitory effect of inorganic phosphate on Dicer-2 processing of blunt-end hairpin RNAs, allowing siRNA production in the presence of inorganic phosphate.

Purification of Microprocessor-Associated Factors.

The Microprocessor complex catalyzes the first step of miRNA biogenesis in the nucleus of mammalian cells. The minimal catalytically active complex is formed by two essential factors, the dsRNA binding protein DGCR8, and the RNase III endonuclease Drosha. Importantly, several co-factors can associate to this complex and modulate the cleavage and binding efficiency of this complex, in a positive or negative manner. Here, we describe a simple method for purification of DGCR8 and Drosha coupled to mass spectrometry or western blot which allows robust identification of unknown associated factors. This approach has recently revealed the presence of a new DGCR8-dependent, Drosha-independent complex involved in RNA turnover.

Innate immune recognition of double-stranded RNA triggers increased expression of NKG2D ligands after virus infection

Self/non-self-discrimination by the innate immune system relies on germline-encoded, non-rearranging receptors expressed by innate immune cells recognizing conserved pathogen-associated molecular patterns. The natural killer group 2D (NKG2D) receptor is a potent immune-activating receptor that binds human genome-encoded ligands, whose expression is negligible in normal tissues, but increased in stress and disease conditions for reasons that are incompletely understood. Here it is not clear how the immune system reconciles receptor binding of self-proteins with self/non-self-discrimination to avoid autoreactivity. We now report that increased expression of NKG2D ligands after virus infection depends on interferon response factors activated by the detection of viral double-stranded RNA by pattern-recognition receptors (RIG-I/MDA-5) and that NKG2D ligand up-regulation can be blocked by the expression of viral dsRNA-binding proteins. Thus, innate immunity-mediated recognition of viral nucleic acids triggers the infected cell to release interferon for NK cell recruitment and to express NKG2D ligands to become more visible to the immune system. Finally, the observation that NKG2D-ligand induction is a consequence of signaling by pattern-recognition receptors that have been selected over evolutionary time to be highly pathogen-specific explains how the risks of autoreactivity in this system are minimized.

HIV-1 NC-induced stress granule assembly and translation arrest are inhibited by the dsRNA binding protein Staufen1.

The nucleocapsid (NC) is an N-terminal protein derived from the HIV-1 Gag precursor polyprotein, pr55Gag. NC possesses key functions at several pivotal stages of viral replication. For example, an interaction between NC and the host double-stranded RNA-binding protein Staufen1 was shown to regulate several steps in the viral replication cycle, such as Gag multimerization and genomic RNA encapsidation. In this work, we observed that the overexpression of NC leads to the induction of stress granule (SG) assembly. NC-mediated SG assembly was unique as it was resistant to the SG blockade imposed by the HIV-1 capsid (CA), as shown in earlier work. NC also reduced host cell mRNA translation, as judged by a puromycylation assay of de novo synthesized proteins, and this was recapitulated in polysome profile analyses. Virus production was also found to be significantly reduced. Finally, Staufen1 expression completely rescued the blockade to NC-mediated SG assembly, global mRNA translation as well as virus production. NC expression also resulted in the phosphorylation of protein kinase R (PKR) and eIF2α, and this was inhibited with Staufen1 coexpression. This work sheds light on an unexpected function of NC in host cell translation. A comprehensive understanding of the molecular mechanisms by which a fine balance of the HIV-1 structural proteins NC and CA act in concert with host proteins such as Staufen1 to modulate the host stress response will aid in the development of new antiviral therapeutics.

Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins.

Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs.

Structural insights into the Middle East respiratory syndrome coronavirus 4a protein and its dsRNA binding mechanism

Middle East respiratory syndrome coronavirus (MERS-CoV) has evolved to navigate through the sophisticated network of a host’s immune system. The immune evasion mechanism including type 1 interferon and protein kinase R-mediated antiviral stress responses has been recently attributed to the involvement of MERS-CoV protein 4a (p4a) that masks the viral dsRNA. However, the structural mechanism of how p4a recognizes and establishes contacts with dsRNA is not well explained. In this study, we report a dynamic mechanism deployed by p4a to engage the viral dsRNA and make it unavailable to the host immune system. Multiple variants of p4a-dsRNA were created and investigated through extensive molecular dynamics procedures to highlight crucial interfacial residues that may be used as potential pharmacophores for future drug development. The structural analysis revealed that p4a exhibits a typical αβββα fold structure, as found in other dsRNA-binding proteins. The α1 helix and the β1-β2 loop play a crucial role in recognizing and establishing contacts with the minor grooves of dsRNA. Further, mutational and binding free energy analyses suggested that in addition to K63 and K67, two other residues, K27 and W45, might also be crucial for p4a-dsRNA stability.

PACT Facilitates RNA-Induced Activation of MDA5 by Promoting MDA5 Oligomerization.

MDA5 is a RIG-I-like cytoplasmic sensor of dsRNA and certain RNA viruses, such as encephalomyocarditis virus, for the initiation of the IFN signaling cascade in the innate antiviral response. The affinity of MDA5 toward dsRNA is low, and its activity becomes optimal in the presence of unknown cellular coactivators. In this article, we report an essential coactivator function of dsRNA-binding protein PACT in mediating the MDA5-dependent type I IFN response. Virus-induced and polyinosinic-polycytidylic acid-induced activation of MDA5 were severely impaired in PACT-knockout cells and attenuated in PACT-knockdown cells, but they were potentiated when PACT was overexpressed. PACT augmented IRF3-dependent type I IFN production subsequent to dsRNA-induced activation of MDA5. In contrast, PACT had no influence on MDA5-mediated activation of NF-κB. PACT required dsRNA interaction for its action on MDA5 and promoted dsRNA-induced oligomerization of MDA5. PACT had little stimulatory effect on MDA5 mutants deficient for oligomerization and filament assembly. PACT colocalized with MDA5 in the cytoplasm and potentiated MDA5 recruitment to the dsRNA ligand. Taken together, these findings suggest that PACT functions as an essential cellular coactivator of RIG-I, as well as MDA5, and it facilitates RNA-induced formation of MDA5 oligomers.

Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies.

The Dicer family of ribonucleases plays a key role in small RNA-based regulatory pathways by generating short dsRNA fragments that modulate expression of endogenous genes, or protect the host from invasive nucleic acids. Beginning with its initial discovery, biochemical characterization of Dicer has provided insight about its catalytic properties. However, a comprehensive understanding of how Dicer's domains contribute to substrate-specific recognition and catalysis is lacking. One reason for this void is the lack of high-resolution structural information for a metazoan Dicer in the apo- or substrate-bound state. Both biochemical and structural studies are facilitated by large amounts of highly purified, active protein, and Dicer enzymes have historically been recalcitrant to overexpression and purification. Here we describe optimized procedures for the large-scale expression of Dicer in baculovirus-infected insect cells. We then outline a three-step protocol for the purification of large amounts (3-4mg of Dicer per liter of insect cell culture) of highly purified and active Dicer protein, suitable for biochemical and structural studies. Our methods are general and are extended to enable overexpression, purification and biochemical characterization of accessory dsRNA binding proteins that interact with Dicer and modulate its catalytic activity.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

The double-stranded RNA binding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans.

Long double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm Caenorhabditis elegans, where the dsRNA-binding protein RDE-4 initiates silencing by recruiting an endonuclease to process long dsRNA into short dsRNA. These short dsRNAs are thought to move between cells because muscle-specific rescue of rde-4 using repetitive transgenes enables silencing in other tissues. Here, we extend this observation using additional promoters, report an inhibitory effect of repetitive transgenes, and discover conditions for cell-autonomous silencing in animals with tissue-specific rescue of rde-4. While expression of rde-4(+) in intestine, hypodermis, or neurons using a repetitive transgene can enable silencing also in unrescued tissues, silencing can be inhibited wihin tissues that express a repetitive transgene. Single-copy transgenes that express rde-4(+) in body-wall muscles or hypodermis, however, enable silencing selectively in the rescued tissue but not in other tissues. These results suggest that silencing by the movement of short dsRNA between cells is not an obligatory feature of feeding RNAi in C. elegans. We speculate that similar control of dsRNA movement could modulate tissue-specific silencing by feeding RNAi in other invertebrates.

The nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same IFN-β antagonizing mechanism: attenuation of PACT-mediated RIG-I/ MDA5 activation.

Coronaviruses (CoVs) are a huge threat to both humans and animals and have evolved elaborate mechanisms to antagonize interferons (IFNs). Nucleocapsid (N) protein is the most abundant viral protein in CoV-infected cells, and has been identified as an innate immunity antagonist in several CoVs, including mouse hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV. However, the underlying molecular mechanism(s) remain unclear. In this study, we found that MHV N protein inhibited Sendai virus and poly(I:C)-induced IFN-β production by targeting a molecule upstream of retinoic acid-induced gene I (RIG-I) and melanoma differentiation gene 5 (MDA5). Further studies showed that both MHV and SARS-CoV N proteins directly interacted with protein activator of protein kinase R (PACT), a cellular dsRNA-binding protein that can bind to RIG-I and MDA5 to activate IFN production. The N–PACT interaction sequestered the association of PACT and RIG-I/MDA5, which in turn inhibited IFN-β production. However, the N proteins from porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV), which are also classified in the order Nidovirales, did not interact and counteract with PACT. Taken together, our present study confirms that both MHV and SARS-CoV N proteins can perturb the function of cellular PACT to circumvent the innate antiviral response. However, this strategy does not appear to be used by all CoVs N proteins.

Live Cell Imaging Reveals the Relocation of dsRNA Binding Proteins Upon Viral Infection

Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins DRB3 and DRB4 are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3, and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRC) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis by T-DNA insertion rendered these plants less able to repress RNA viral replication. We propose a model for the early stages of virus defense in which DRB2, DRB3, and DRB5 are invasion sensors that relocate to nascent VRC, where they bind to viral RNA and inhibit virus replication.

New insights into reovirus evolution: implications from a newly characterized mycoreovirus.

We performed molecular cloning and complete genome sequencing of a novel mycoreovirus, Sclerotinia sclerotiorumreovirus 1 (SsReV1), isolated from an isolate of the phytopathogenic fungus Sclerotinia sclerotiorum. SsReV1 has a genome of 28 055 bp and is composed of 11 double-stranded RNA segments. With a combination of unique molecular features, virion shape and composition, and phylogenetic analysis, SsReV1 is significantly distinct from all known reoviruses and defines a novel genus in the family Reoviridae. Interestingly, two conserved domains, double-stranded RNA binding motif (dsRBM, Pfam 00 035) and reovirus sigma C capsid protein (Reo_σC, pfam04 582), were identified in the genome of SsReV1, which are widespread in diverse virus lineages. Sequence comparison and phylogenetic analysis revealed that multiple cross-family horizontal gene transfer (HGT) events could occur between reoviruses and double-stranded DNA viruses, single-stranded RNA viruses and even cellular organisms. Interestingly, the dsRBM of SsReV1 was phylogenetically related to dsRNA-binding proteins of some insects, but not reoviruses. These results indicated that SsReV1 is a new taxonomic representative in Reoviridae, which provides new insights into the diversity and global ecology of reoviruses and other segmented double-stranded RNA viruses. More importantly, the present results provided evidence indicating that reoviruses indeed have HGT events with other virus lineages on a large scale and that HGT may serve as an important driving factor that plays a key role in the evolution of reoviruses.

Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates

MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.

A complex of Arabidopsis DRB proteins can impair dsRNA processing.

Small RNAs play an important role in regulating gene expression through transcriptional and post-transcriptional gene silencing. Biogenesis of small RNAs from longer double-stranded (ds) RNA requires the activity of dicer-like ribonucleases (DCLs), which in plants are aided by dsRNA binding proteins (DRBs). To gain insight into this pathway in the model plant Arabidopsis, we searched for interactors of DRB4 by immunoprecipitation followed by mass spectrometry-based fingerprinting and discovered DRB7.1. This interaction, verified by reciprocal coimmunoprecipitation and bimolecular fluorescence complementation, colocalizes with markers of cytoplasmic siRNA bodies and nuclear dicing bodies. In vitro experiments using tobacco BY-2 cell lysate (BYL) revealed that the complex of DRB7.1/DRB4 impairs cleavage of diverse dsRNA substrates into 24-nucleotide (nt) small interfering (si) RNAs, an action performed by DCL3. DRB7.1 also negates the action of DRB4 in enhancing accumulation of 21-nt siRNAs produced by DCL4. Overexpression of DRB7.1 in Arabidopsis altered accumulation of siRNAs in a manner reminiscent of drb4 mutant plants, suggesting that DRB7.1 can antagonize the function of DRB4 in siRNA accumulation in vivo as well as in vitro. Specifically, enhanced accumulation of siRNAs from an endogenous inverted repeat correlated with enhanced DNA methylation, suggesting a biological impact for DRB7.1 in regulating epigenetic marks. We further demonstrate that RNase three-like (RTL) proteins RTL1 and RTL2 cleave dsRNA when expressed in BYL, and that this activity is impaired by DRB7.1/DRB4. Investigating the DRB7.1–DRB4 interaction thus revealed that a complex of DRB proteins can antagonize, rather than promote, RNase III activity and production of siRNAs in plants.

MyxomaVirusdsRNABindingProteinM029 InhibitstheTypeIIFN-InducedAntiviralStateina HighlySpecies-SpecificFashion.

Myxoma virus (MYXV) is a Leporipoxvirus that possesses a specific rabbit-restricted host tropism but exhibits a much broader cellular host range in cultured cells. MYXV is able to efficiently block all aspects of the type I interferon (IFN)-induced antiviral state in rabbit cells, partially in human cells and very poorly in mouse cells. The mechanism(s) of this species-specific inhibition of type I IFN-induced antiviral state is not well understood. Here we demonstrate that MYXV encoded protein M029, a truncated relative of the vaccinia virus (VACV) E3 double-stranded RNA (dsRNA) binding protein that inhibits protein kinase R (PKR), can also antagonize the type I IFN-induced antiviral state in a highly species-specific manner. In cells pre-treated with type I IFN prior to infection, MYXV exploits M029 to overcome the induced antiviral state completely in rabbit cells, partially in human cells, but not at all in mouse cells. However, in cells pre-infected with MYXV, IFN-induced signaling is fully inhibited even in the absence of M029 in cells from all three species, suggesting that other MYXV protein(s) apart from M029 block IFN signaling in a species-independent manner. We also show that the antiviral state induced in rabbit, human or mouse cells by type I IFN can inhibit M029-knockout MYXV even when PKR is genetically knocked-out, suggesting that M029 targets other host proteins for this antiviral state inhibition. Thus, the MYXV dsRNA binding protein M029 not only antagonizes PKR from multiple species but also blocks the type I IFN antiviral state independently of PKR in a highly species-specific fashion.

Circular RNAs: A novel type of non-coding RNA and their potential implications in antiviral immunity.

Circular RNAs (circRNAs), a novel type of non-coding RNAs (ncRNAs), are ubiquitously expressed in eukaryotic cells during post-transcriptional processes. Unlike linear RNAs, circRNAs form covalent-closed continuous loops without 5' to 3' polarities and poly (A) tails. With advances in high-throughput sequencing technology, numerous circRNAs have been identified in plants, animals and humans. Notably, circRNAs display cell-type, tissue-type and developmental-stage specific expression patterns in eukaryotic transcriptome, which reveals their significant regulatory functions in gene expression. More importantly, circRNAs serve as microRNA (miRNA) sponges and crucial regulators of gene expression. Additionally, circRNAs modulate pre-mRNA alternative splicing and possess protein-coding capacity. CircRNAs exhibit altered expression under pathological conditions and are strongly associated with the development of various human diseases. Interestingly, circRNAs can also induce antiviral immune responses. A recent study found that the delivery of circRNAs generated in vitro activates RIG-I-mediated innate immune responses and provides protection against viral infection. The antiviral dsRNA-binding proteins, NF90/NF110, act as key regulators in circRNA biogenesis. NF90/NF110 are also functional in inhibiting viral replication through binding to viral mRNAs. In this review, we provide a comprehensive overview on the classification, biogenesis and functions of circRNAs. We also discuss the critical role of circRNAs in eliciting antiviral immunity, providing evidence for the potential implications of circRNAs in antiviral therapies.

A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing.

In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.