Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

Intracellular recognition of non‐self and also self‐nucleic acids can result in the initiation of potent pro‐inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2′–5′), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP‐1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS–STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.     

Perception of double-stranded RNA in plant antiviral immunity

RNA silencing and antiviral pattern-triggered immunity (PTI) both rely on recognition of double-stranded (ds)RNAs as defence-inducing signals. While dsRNA recognition by dicer-like proteins during antiviral RNA silencing is thoroughly investigated, the molecular mechanisms involved in dsRNA perception leading to antiviral PTI are just about to be untangled. Parallels to antimicrobial PTI thereby only partially facilitate our view on antiviral PTI. PTI against microbial pathogens involves plasma membrane bound receptors; however, dsRNAs produced during virus infection occur intracellularly. Hence, how dsRNA may be perceived during this immune response is still an open question. In this short review, we describe recent discoveries in PTI signalling upon sensing of microbial patterns and endogenous ‘danger’ molecules with emphasis on immune signalling-associated subcellular trafficking processes in plants. Based on these studies, we develop different scenarios how dsRNAs could be sensed during antiviral PTI.     

piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline

Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish "self" from "nonself" nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of "self" and "nonself" RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.     

Polyinosinic acid is a ligand for toll-like receptor 3

Innate immune responses are critical in controlling viral infections. Viral proteins and nucleic acids have been shown to be recognized by pattern recognition receptors of the Toll-like receptor (TLR) family, triggering downstream signaling cascades that lead to cellular activation and cytokine production. Viral DNA is sensed by TLR9, and TLRs 3, 7, and 8 have been implicated in innate responses to RNA viruses by virtue of their ability to sense double-stranded (ds) RNA (TLR3) or single-stranded RNA (murine TLR7 and human TLR8). Viral and synthetic dsRNAs have also been shown to be a potent adjuvant, promoting enhanced adaptive immune responses, and this property is also dependent on their recognition by TLR3. It has recently been shown that mRNA that is largely single-stranded is a ligand for TLR3. Here we have investigated the ability of single-stranded homopolymeric nucleic acids to induce innate responses by murine immune cells. We show for the first time that polyinosinic acid (poly(I)) activates B lymphocytes, dendritic cells, and macrophages and that these responses are dependent on the expression of both TLR3 and the adaptor molecule, Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF). We therefore conclude that TLR3 is able to sense both single-stranded RNA and dsRNA.     

Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

Abstract Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells, although its gene silencing efficiency is lower than that of exogenously introduced double‐stranded RNAs (dsRNAs). To solve this problem, we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA (siRNA). Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm, but were not effectively diced into siRNAs. Interestingly, RNAi with hairpin dsRNAs from genome‐integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase III, although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.     

Interferon-beta induction through toll-like receptor 3 depends on double-stranded RNA structure

Type I interferons (IFN-alpha/beta) play an essential role in both innate and adaptive antiviral immune responses. IFN- beta is produced by fibroblasts and myeloid dendritic cells (DCs) upon viral infection or in response to doublestranded RNA (dsRNA). Several intracellular molecules having a dsRNA-binding motif such as dsRNA-dependent protein kinase recognize dsRNA in a sequence-independent manner and induce antiviral innate responses. Toll-like receptor (TLR) 3, a member of TLR family proteins, recognizes extracellular dsRNA and activates NF- kappaB and the IFN-beta promoter leading to the induction of IFN-beta production. Here we analyzed the dsRNA structure capable of inducing TLR3-mediated IFN-beta production using various synthetic RNA duplexes. In contrast to the recognition of dsRNA by intracellular molecules, TLR3 preferentially recognizes polyriboinocinic:polyribocytidylic acid (poly(I:C)) rather than synthetic virus-derived dsRNAs. 2'-O-methyl or 2'-fluoro modification of cytidylic acid abolished the IFN-beta-inducing ability of the poly(I:C) duplex, and these modified dsRNAs inhibited poly(I:C)-induced TLR3-mediated IFN-beta production by fibroblasts and DCs. In addition, poly(dI:dC), a non-IFN inducer, also blocked poly(I:C)-induced IFN-beta induction. Since TLR3 is localized in the intracellular compartment of DCs where signaling occurs, modified dsRNAs may compete with poly(I:C) for binding to the cell-surface receptor that transfers dsRNA into TLR3-enriched vesicles. Thus, TLR3 recognizes a unique dsRNA structure that largely differs from those recognized by other dsRNA-binding proteins.     

新药原料——低毒性、抗病毒、抗肿瘤失配双链核糖核酸的研究进展

新药原料--低毒性、抗病毒、抗肿瘤失配双链核糖核酸的研究进展聂实践胡冬琴赵红罗薇（北京市营养源研究所真菌工程实验室）前言近年来,全球性病毒性疾病在不断增加,但对抗病毒药物的研究却进展缓慢。直至今日由病毒引起的恶性疾病（如恶性肿瘤,ＡＩＤＳ病等）仍无法医治,这已成为当今严重的社会问题。据统计约６０％的流行性传染病都是由病毒感染引起的,细菌感染仅占１５％。而目前世界各国批准使用的合成抗病毒药物仅...     

Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys

Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.     

Unusual inheritance of evolutionarily-related double-stranded RNAs in interspecific hybrid between rice plants Oryza sativa and Oryza rufipogon

Endogenous, 14 kb double-stranded RNAs (dsRNAs) have been found in two ecospecies of cultivated rice (temperate japonica rice and tropical japonica rice, Oryza sativa L.) and in wild rice (O. rufipogon, an ancestor of O. sativa). A comparison of the nucleotide and deduced amino acid sequences of the core regions of the RNA-dependent RNA polymerase domains found in these three dsRNAs suggested that these dsRNAs probably evolved independently within each host plant from a common ancestor. These dsRNAs were introduced into F1 hybrids by crossing cultivated rice and wild rice. Unusual cytoplasmic inheritance of these dsRNAs was observed in some F1 hybrids; the evolutionarily related dsRNAs were incompatible for each other, and the resident dsRNA of an egg cell from cultivated rice was excluded by the incoming dsRNA of a pollen cell from wild rice. Coexisting dsRNAs in the F1 hybrids segregated away from each other in the F2 plants. However, the total amount of these dsRNAs in the host cells remained constant (ca. 100 copies/cell). The stringent regulation of the dsRNA copy number may be responsible for their unusual inheritance.     

A Rapid Method for Sequencing the 5'- and 3'-Termini of Double-Stranded RNA Viral Templates using RLM-RACE

A protocol is described that permits rapid, inexpensive and reliable synthesis and amplification of 5′‐ and 3′‐terminal cDNAs using viral double‐stranded (ds) RNA templates. The method of RNA ligase‐mediated amplification of cDNA ends for amplification of single‐stranded RNAs ( Liu and Gorovsky, 1993 ) was adapted for dsRNAs to generate the amplicons. These amplicons can be easily cloned and sequenced to complete the sequencing of 5′‐ and 3′‐termini of any particular virus genome. The method is of great utility and can be used on viral genomic dsRNA or viral dsRNA replicative forms when the virion RNA is unavailable.     

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occurring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNA-DNA hybrids was observed. The apparent affinity of the antibodies to short (less than or equal to 11 bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm- values of GRV dsRNA-3.     

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

RNA interference (RNAi) elicited by long double‐stranded (ds) or base‐paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence‐specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN‐responsive cells with type I IFN. Notably, transfection with long dsRNA specifically vaccinates IFN‐deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.     

Small RNAs, RNAi and the Inheritance of Gene Silencing in Caenorhabditis elegans

Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics, e.g., unpaired DNA or abnormal double-stranded RNA. Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids. Furthermore, they have developed ways to remember this exposure to invaders and transmit the experience to their descendants. The mechanism underlying this inheritance has remained elusive. Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing. Small regulatory RNAs play a variety of crucial roles in organisms, including gene regulation, developmental timing, antiviral defense, and genome integrity, via a process termed as RNA interference (RNAi). Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing. Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations. Notably, these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations. Thus, RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.     

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae

Abstract Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.