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.     

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.     

RNA structural motif recognition based on least-squares distance

RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.     

Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.     

RNA干扰

RNA干扰(RNAinterference,RNAi)是一种古老的生物抗病毒机制,能介导序列特异性的mRNA降解,是基因功能研究和蛋白组学的有效工具,在药物靶基因的筛选、抗病毒、肿瘤基因治疗等领域有很好的发展前景。     

Omnipotent RNA

The capability of polyribonucleotide chains to form unique, compactly folded structures is considered the basis for diverse non-genetic functions of RNA, including the function of recognition of various ligands and the catalytic function. Together with well-known genetic functions of RNA – coding and complementary replication – this has led to the concept of the functional omnipotence of RNA and the hypothesis that an ancient RNA world supposedly preceded the contemporary DNA–RNA–protein life. It is proposed that the Woese universal precursor in the ancient RNA world could be a cell-free community of mixed RNA colonies growing and multiplying on solid surfaces.     

Characterization of the double-stranded RNA responses in human liver progenitor cells

Human HepaRG cells are liver progenitors which possess hepatocyte-like functionality. We investigated the effects of double-stranded (ds) RNA on interferon (IFN)-β and chemokine (CK) expression in these cells. By microarray and ELISA, we showed strong induction of CXCL10 and interleulin (IL)-8 besides IFN-β and other CK ligands. RNA interference directed silencing of TLR3, RIG-I, IRF3, NFκB or MAP kinases (p38, ERK, JNK) was carried out. Knockdown of all these molecules, except ERK and JNK, blocked IFN-β production. Both TLR3 and RIG-I are required for CXCL10 expression. Silencing of TLR3 completely impaired the IL-8 expression. dsRNA-conditioned medium from HepaRG cells exerted a drastic antiviral effect in HCV replicons, and in the JFH-1-based HCV production cell culture system. The IFN-β knockdown in HepaRG cells removed this antiviral effect but did not enhance their capacity to initiate HCV RNA replication. We conclude that dsRNA induces antiviral and pro-inflammatory status in HepaRG cells.     

Structure of the RNA Binding Domain of a DEAD-Box Helicase Bound to Its Ribosomal RNA Target Reveals a Novel Mode of Recognition by an RNA Recognition Motif

DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 Å resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairpin 92 interact with the RBD, including the guanosine base of G2553, which forms three hydrogen bonds with the peptide backbone. A G2553U mutation reduces the RNA binding affinity by 2 orders of magnitude, confirming that G2553 is a sequence specificity determinant in RNA binding. Binding of the RBD to 23S rRNA in the late stages of ribosome subunit maturation would position the ATP-binding duplex destabilization fragment of the protein for interaction with rRNA in the peptidyl transferase cleft of the subunit, allowing it to “melt out” unstable secondary structures and allow proper folding.     

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3′ to inosine in single-strand RNA, at a low reaction temperature of 20–25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.     

Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-stranded RNA

3′ repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent K_m of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1.     

昆虫RNA沉默抗病毒机制研究进展

RNA沉默是昆虫用来抵御病毒入侵的一种普遍而又进化保守的防御机制, 而昆虫病毒也会相应地编码沉默抑制子来破坏宿主的防御功能。本文主要结合果蝇的相关研究成果对昆虫RNA沉默抗病毒机制、 RNA沉默抑制子的作用特征及宿主与病毒的共进化关系做一综述。研究表明, 由小干扰RNA (small interfering RNAs, siRNA)介导的RNA干扰在果蝇抗病毒防御机制中发挥重要作用。果蝇中Dicer-2（Dcr-2）, argonaute-2（AGO2）和双链RNA结合蛋白R2D2是siRNA干扰途径中的3个关键组分, 这3个基因的缺失或突变会显著提高果蝇对RNA病毒的感受性。此外, 果蝇中还鉴定了其他与RNA干扰密切相关的基因, 如vasa intronic gene, aubergine, armitage, rm62 和piwi, 它们在抗病毒感染中同样发挥重要作用。果蝇病毒中已鉴定出3种RNA沉默病毒抑制子（viral suppressors of RNAi, VSRs）, 分别为果蝇FHV病毒沉默抑制子FHV-B2、 果蝇C病毒沉默抑制子DCV-1A及果蝇CrPV病毒沉默抑制子CrPV-1A。FHV-B2和DCV-1A通过与dsRNA或siRNA结合抑制RNA沉默, 而CrPV-1A通过与AGO2结合阻止RISC的形成抑制RNA沉默。在漫长的进化过程中, 病毒和宿主相互博弈, 协同进化。昆虫抗病毒沉默途径中的关键组分通过保持持续和快速进化来对抗高度变异的VSRs。     

Structures of RNA Complexes with the Escherichia coli RNA Pyrophosphohydrolase RppH Unveil the Basis for Specific 5′-End-dependent mRNA Decay

5′-End-dependent RNA degradation impacts virulence, stress responses, and DNA repair in bacteria by controlling the decay of hundreds of mRNAs. The RNA pyrophosphohydrolase RppH, a member of the Nudix hydrolase superfamily, triggers this degradation pathway by removing pyrophosphate from the triphosphorylated RNA 5′ terminus. Here, we report the x-ray structures of Escherichia coli RppH (EcRppH) in apo- and RNA-bound forms. These structures show distinct conformations of EcRppH·RNA complexes on the catalytic pathway and suggest a common catalytic mechanism for Nudix hydrolases. EcRppH interacts with RNA by a bipartite mechanism involving specific recognition of the 5′-terminal triphosphate and the second nucleotide, thus enabling discrimination against mononucleotides as substrates. The structures also reveal the molecular basis for the preference of the enzyme for RNA substrates bearing guanine in the second position by identifying a protein cleft in which guanine interacts with EcRppH side chains via cation-π contacts and hydrogen bonds. These interactions explain the modest specificity of EcRppH at the 5′ terminus and distinguish the enzyme from the highly selective RppH present in Bacillus subtilis. The divergent means by which RNA is recognized by these two functionally and structurally analogous enzymes have important implications for mRNA decay and the regulation of protein biosynthesis in bacteria.     

Specificity and Evolutionary Conservation of the Escherichia coli RNA Pyrophosphohydrolase RppH

Bacterial RNA degradation often begins with conversion of the 5′-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5′-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5′ end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5′ terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution.