Viroid RNA Research Articles

Direct visualization of the native structure of viroid RNAs at single-molecule resolution by atomic force microscopy

ABSTRACTViroids are small infectious, non-protein-coding circular RNAs that replicate independently and, in some cases, incite diseases in plants. They are classified into two families: Pospiviroidae, composed of species that have a central conserved region (CCR) and replicate in the cell nucleus, and Avsunviroidae, containing species that lack a CCR and whose multimeric replicative intermediates of either polarity generated in plastids self-cleave through hammerhead ribozymes. The compact, rod-like or branched, secondary structures of viroid RNAs have been predicted by RNA folding algorithms and further examined using different in vitro and in vivo experimental techniques. However, direct data about their native tertiary structure remain scarce. Here we have applied atomic force microscopy (AFM) to image at single-molecule resolution different variant RNAs of three representative viroids: potato spindle tuber viroid (PSTVd, family Pospiviroidae), peach latent mosaic viroid and eggplant latent viroid (PLMVd and ELVd, family Avsunviroidae). Our results provide a direct visualization of their native, three-dimensional conformations at 0 and 4 mM Mg2+ and highlight the role that some elements of tertiary structure play in their stabilization. The AFM images show that addition of 4 mM Mg2+ to the folding buffer results in a size contraction in PSTVd and ELVd, as well as in PLMVd when the kissing-loop interaction that stabilizes its 3D structure is preserved.

Viroid research and its significance for RNA technology and basic biochemistry.

Viroids were described 47 years ago as the smallest RNA molecules capable of infecting plants and autonomously self-replicating without an encoded protein. Work on viroids initiated the development of a number of innovative methods. Novel chromatographic and gelelectrophoretic methods were developed for the purification and characterization of viroids; these methods were later used in molecular biology, gene technology and in prion research. Theoretical and experimental studies of RNA folding demonstrated the general biological importance of metastable structures, and nuclear magnetic resonance spectroscopy of viroid RNA showed the partially covalent nature of hydrogen bonds in biological macromolecules. RNA biochemistry and molecular biology profited from viroid research, such as in the detection of RNA as template of DNA-dependent polymerases and in mechanisms of gene silencing. Viroids, the first circular RNA detected in nature, are important for studies on the much wider spectrum of circular RNAs and other non-coding RNAs.

Enrichment of midsized RNAs with manganese chloride precipitation

Enrichment of specific RNAs is important for RNA analysis. MnCl2 has been used previously to enrich viroid RNA fractions from total RNA from infected plants. We have expanded this method to show that MnCl2 can enrich single-stranded as well as structured RNAs of 450 nt and below from a total RNA preparation. We have applied this method to map the transcription start sites of a PSTVd transcript from total RNA from yeast under conditions where the RNA was previously undetectable.

Mutational Analysis of Eggplant Latent Viroid RNA Circularization by the Eggplant tRNA Ligase in Escherichia coli.

Eggplant latent viroid (ELVd) is a relatively small non-coding circular RNA that induces asymptomatic infections in eggplants (Solanum melongena L.). Like other viroid species that belong to the family Avsunviroidae, ELVd contains hammerhead ribozymes in the strands of both polarities that self-cleave RNAs producing terminal 5′-hydroxyl and 2′,3′-cyclic phosphodiester groups. Available experimental data indicate that ELVd replicates in the chloroplasts of infected cells through a symmetric rolling-circle mechanism, in which RNA circularization is catalyzed by the chloroplastic isoform of the tRNA ligase. In this work, a mutational analysis was performed to gain insight into the sequence and structural requirements of the tRNA ligase-mediated circularization of ELVd RNAs. In the predicted minimum free energy conformation of the monomeric linear ELVd RNA intermediate of plus (+) polarity, the ligation site is located in the lower part of an opened internal loop, which is present in a quasi-rod-like structure that occupies the center of the molecule. The mutations analyzed herein consisted of punctual nucleotide substitutions and deletions surrounding the ligation site on the upper and lower strands of the ELVd quasi-double-stranded structure. Computational predictions of the mutated ELVd conformations indicated different degrees of distortions compared to the minimum free energy conformation of the wild-type ELVd linear monomer of + polarity. When these mutant RNAs were expressed in Escherichia coli, they were all circularized by the eggplant tRNA ligase with approximately the same efficiency as the wild-type ELVd, except for those that directly affected the ribozyme domain. These results suggest that the viroid ribozyme domains, in addition to self-cleavage, are also involved in the tRNA ligase-mediated circularization of the monomeric linear replication intermediates.

Allelic RNA Motifs in Regulating Systemic Trafficking of Potato Spindle Tuber Viroid.

Intercellular RNA trafficking has been shown as a widely-existing phenomenon that has significant functions in many aspects of biology. Viroids, circular noncoding RNAs that cause plant diseases, have been a model to dissect the role of RNA structural motifs in regulating intercellular RNA trafficking in plants. Recent studies on potato spindle tuber viroid (PSTVd) showed that the RNA motif loop 19 is important for PSTVd to spread from palisade to spongy mesophyll in infected leaves. Here, we performed saturated mutational analysis to uncover all possible functional variants of loop 19 and exploit this data to pinpoint to a three-dimensional structural model of this motif. Interestingly, we found that two distinct structural motifs can replace loop 19 and retain the systemic trafficking capacity. One of the alternative structures rapidly emerged from the inoculation using a loop 19 abolished mutant that is not capable of systemic trafficking. Our observation indicates the flexibility of multiple structural arrangements interchangeably exerting similar function at a particular RNA locus. Taken together, this study deepens the understanding of RNA structural motifs-regulated viroid RNA trafficking, which has broad implications for studying RNA intercellular trafficking as well.

RNA 3-dimensional structural motifs as a critical constraint of viroid RNA evolution.

Viroids are circular noncoding RNAs infecting plants [1, 2]. During infection, viroids, like RNA viruses, generate swarms of sequence variants called quasispecies [3, 4]. Viroids in Avsunviroidae family replicate in chloroplasts and display the highest mutation rates among all living entities [5]. Viroids in Pospiviroidae family replicate in the nucleus with a relatively lower mutation rate resembling some RNA viruses [6]. Those sequence variants generated during replication are described by the concept of sequence space, which harnesses a geometric representation to illustrate genetic similarities via physical distances. Given the high mutation rate and fast propagation, viroid RNAs have a potentially large sequence space for the evolution of new variants. However, in reality, they use only a small fraction of this space. Constraints of viral sequence space may include genome size, replication fidelity, error thresholds, host or tissue tropism, etc. These factors have been nicely reviewed elsewhere [3, 7, 8] and are not the focus of this Pearl. In addition, RNA secondary structures have been considered, though not adequately, as a constraint factor [8]. Viroids, in contrast to viruses, entirely rely on their RNA structural motifs for function due to their noncoding nature, which offers insights into their capacity to explore regions of sequence space influenced by RNA structures. Here, we describe that 3-dimensional (3D) structural motifs formed by non–Watson-Crick (non-WC) base pairs in viroid RNAs act as a critical constraint for the sequence space of viroid genome evolution. This constraint operates because RNA 3D motifs can play crucial roles by mediating (1) RNA–RNA interactions for the folding of a part or a whole of RNA into a distinct tertiary conformation and (2) RNA–protein interactions. Therefore, mutations in a 3D motif that do not disrupt the structure and function will be retained in the population, whereas mutations that disrupt the 3D structures of motifs, and consequently the function, will be lost.

Circular RNA shifts the miRNA binding panel and takes a role in hepatocellular carcinoma progression

Circular RNAs (circRNAs) are a novel type of RNA molecule that covalently joint its 5' and 3' end together and form a closed single strand annular. CircRNAs were first reported by Sanger et al . as a plant viroid RNA in 1976 (1). In decades, circRNAs were rarely investigated mainly because they are usually expressed at a relative low level.

Processing of Potato Spindle Tuber Viroid RNAs in Yeast, a Nonconventional Host.

Potato spindle tuber viroid (PSTVd) is a circular, single-stranded, noncoding RNA plant pathogen that is a useful model for studying the processing of noncoding RNA in eukaryotes. Infective PSTVd circles are replicated via an asymmetric rolling circle mechanism to form linear multimeric RNAs. An endonuclease cleaves these into monomers, and a ligase seals these into mature circles. All eukaryotes may have such enzymes for processing noncoding RNA. As a test, we investigated the processing of three PSTVd RNA constructs in the yeast Saccharomyces cerevisiae Of these, only one form, a construct that adopts a previously described tetraloop-containing conformation (TL), produces circles. TL has 16 nucleotides of the 3' end duplicated at the 5' end and a 3' end produced by self-cleavage of a delta ribozyme. The other two constructs, an exact monomer flanked by ribozymes and a trihelix-forming RNA with requisite 5' and 3' duplications, do not produce circles. The TL circles contain nonnative nucleotides resulting from the 3' end created by the ribozyme and the 5' end created from an endolytic cleavage by yeast at a site distinct from where potato enzymes cut these RNAs. RNAs from all three transcripts are cleaved in places not on path for circle formation, likely representing RNA decay. We propose that these constructs fold into distinct RNA structures that interact differently with host cell RNA metabolism enzymes, resulting in various susceptibilities to degradation versus processing. We conclude that PSTVd RNA is opportunistic and may use different processing pathways in different hosts.IMPORTANCE In higher eukaryotes, the majority of transcribed RNAs do not encode proteins. These noncoding RNAs are responsible for mRNA regulation, control of the expression of regulatory microRNAs, sensing of changes in the environment by use of riboswitches (RNAs that change shape in response to environmental signals), catalysis, and more roles that are still being uncovered. Some of these functions may be remnants from the RNA world and, as such, would be part of the evolutionary past of all forms of modern life. Viroids are noncoding RNAs that can cause disease in plants. Since they encode no proteins, they depend on their own RNA and on host proteins for replication and pathogenicity. It is likely that viroids hijack critical host RNA pathways for processing the host's own noncoding RNA. These pathways are still unknown. Elucidating these pathways should reveal new biological functions of noncoding RNA.

Sensitivity enhancement of graphene/zinc oxide nanocomposite-based electrochemical impedance genosensor for single stranded RNA detection

An efficient electrochemical impedance genosensing platform has been constructed based on graphene/zinc oxide nanocomposite produced via a facile and green approach. Highly pristine graphene was synthesised from graphite through liquid phase sonication and then mixed with zinc acetate hexahydrate for the synthesis of graphene/zinc oxide nanocomposite by solvothermal growth. The as-synthesised graphene/zinc oxide nanocomposite was characterised with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and X-ray diffractometry (XRD) to evaluate its morphology, crystallinity, composition and purity. An amino-modified single stranded DNA oligonucleotide probe synthesised based on complementary Coconut Cadang-Cadang Viroid (CCCVd) RNA sequence, was covalently bonded onto the surface of graphene/zinc oxide nanocomposite by the bio-linker 1-pyrenebutyric acid N-hydroxysuccinimide ester. The hybridisation events were monitored by electrochemical impedance spectroscopy (EIS). Under optimised sensing conditions, the single stranded CCCVd RNA oligonucleotide target could be quantified in a wide range of 1.0×10–11M to 1.0×10−6 with good linearity (R =0.9927), high sensitivity with low detection limit of 4.3×10–12M. Differential pulse voltammetry (DPV) was also performed for the estimation of nucleic acid density on the graphene/zinc oxide nanocomposite-modified sensing platform. The current work demonstrates an important advancement towards the development of a sensitive detection assay for various diseases involving RNA agents such as CCCVd in the future.

Functional analysis of a viroid RNA motif mediating cell-to-cell movement in Nicotiana benthamiana.

Cell-to-cell trafficking through different cellular layers is a key process for various RNAs including those of plant viruses and viroids, but the regulatory mechanisms involved are still not fully elucidated and good model systems are important. Here, we analyse the function of a simple RNA motif (termed 'loop19') in potato spindle tuber viroid (PSTVd) which is required for trafficking in Nicotiana benthamiana leaves. Northern blotting, reverse transcriptase PCR (RT-PCR) and in situ hybridization analyses demonstrated that unlike wild-type PSTVd, which was present in the nuclei in all cell types, the trafficking-defective loop19 mutants were visible only in the nuclei of upper epidermal and palisade mesophyll cells, which shows that PSTVd loop19 plays a role in mediating RNA trafficking from palisade to spongy mesophyll cells in N.benthamiana leaves. Our findings and approaches have broad implications for studying the RNA motifs mediating trafficking of RNAs across specific cellular boundaries in other biological systems.

Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A “naked” rod-like conformation similar but not identical to that observed in vitro

ABSTRACTWith a minimal (250–400 nt), non-protein-coding, circular RNA genome, viroids rely on sequence/structural motifs for replication and colonization of their host plants. These motifs are embedded in a compact secondary structure whose elucidation is crucial to understand how they function. Viroid RNA structure has been tackled in silico with algorithms searching for the conformation of minimal free energy, and in vitro by probing in solution with RNases, dimethyl sulphate and bisulphite, and with selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), which interrogates the RNA backbone at single-nucleotide resolution. However, in vivo approaches at that resolution have not been assayed. Here, after confirming by 3 termodynamics-based predictions and by in vitro SHAPE that the secondary structure adopted by the infectious monomeric circular (+) RNA of potato spindle tuber viroid (PSTVd) is a rod-like conformation with double-stranded segments flanked by loops, we have probed it in vivo with a SHAPE modification. We provide direct evidence that a similar, but not identical, rod-like conformation exists in PSTVd-infected leaves of Nicotiana benthamiana, verifying the long-standing view that this RNA accumulates in planta as a “naked” form rather than tightly associated with protecting host proteins. However, certain nucleotides of the central conserved region, including some of the loop E involved in key functions such as replication, are more SHAPE-reactive in vitro than in vivo. This difference is most likely due to interactions with proteins mediating some of these functions, or to structural changes promoted by other factors of the in vivo habitat.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

Callose Deposition in Plasmodesmata and Viroid Invasion of the Shoot Apical Meristem.

Replication of Avocado Sunblotch Viroid in the Cyanobacterium Nostoc Sp. PCC 7120

Viroids are small infectious RNA molecules that replicate in plants via RNA-RNA replication processes. The molecular mechanism responsible for this replication has attracted great interest, and studies on this topic have yielded interesting biological findings on the processes in which RNA is involved. Viroids belonging to the Avsunviroidae family replicate in the chloroplasts of infected hosts. It has by now been established that chloroplasts and cyanobacteria share a common have ancestor. In view of this phylogenetic relationship, we investigated whether a member of the Avsunviroidae family could be replicated in a cyanobacterium. The results obtained here show that Avocado Sunblotch Viroid (ASBVd) RNA is able to replicate in the filamentous cyanobacterium Nostoc PCC 7120. Indeed, Northern blot hybridization showed that linear forms of “minus” polarity were detected in RNA extracted from Nostoc cells expressing ASBVd dimers of positive “polarity”, and that linear forms of “negative” polarity were detected in RNA extracted from Nostoc cells expressing ASBVd dimers of “positive” polarity. ASBVd replication does not impair the growth of Nostoc. These results provide the first evidence that a prokaryotic cell possesses all the machinery required to sustain the process of RNA-RNA replication. The data obtained here are of great importance, since they might shed light on the evolution of the cellular factors on which RNA replication processes depend.

Human Endosymbiotic Archaea, Retroviral Resistance and Emerging Viral Pandemics: The Crossing of Species Barrier and New Viruses

Human Endosymbiotic Archaea, Retroviral Resistance and Emerging Viral Pandemics: The Crossing of Species Barrier and New Viruses

Apple scar skin viroid naked RNA is actively transmitted by the whitefly Trialeurodes vaporariorum

Nucleic acid transfer between plants is a phenomenon which is likely to occur in many ways in nature. We report here the active transmission of Apple scar skin viroid (ASSVd) naked ssRNA species by the whitefly Trialeurodes vaporariorum (Tv). Not only the viroid RNA, its DNA form was also identified from the insect. The viroid transfer efficiency was enhanced with the help of Cucumis sativus Phloem protein 2 (CsPP2), a plant protein known to translocate viroid RNAs. This PP2/ASSVd complex is stably present in the viroid infected cucumber plants, as was identified with the help of immunological reaction. As viroid-like secondary structures are found in some plant RNAs, and PP2 is known to bind and translocate several RNAs, the results have huge implications in transfer of these RNA species between plants visited by the whitefly.

A Codon-Optimized Nucleic Acid Hydrolyzing Single-Chain Antibody Confers Resistance to Chrysanthemums Against Chrysanthemum Stunt Viroid Infection

Chrysanthemum stunt viroid (CSVd), the smallest plant pathogen known to infect chrysanthemums, is a single-stranded circular RNA viroid that induces stunting that results in an overall height reduction of 30–50 % in mature plants. A catalytic single-chain variable antibody, 3D8 scFv, which exhibits intrinsic DNase and RNase activities, was expressed in chrysanthemums to generate transgenic plant resistance to CSVd infection. Moreover, a codon-optimized version of the 3D8 scFv gene for chrysanthemums was also transformed into plants; these codon-optimized transgenic chrysanthemums expressed twice as much 3D8 scFv and displayed 60 % more resistance to CSVd infection, compared with transgenic chrysanthemums harboring the original 3D8 scFv gene. CSVd challenge experiments with codon-optimized and original 3D8 scFv-transgenic chrysanthemums showed that CSVd in newly produced leaves of both codon-optimized and original 3D8 scFv-transgenic plants was not detected by RT-PCR. This is the first report describing the development of a CSVd-resistant chrysanthemum harboring a catalytic single-chain antibody, 3D8 scFv, which has intrinsic RNase activity.

Development of a duplex one-step RT-qPCR assay for the simultaneous detection of Apple scar skin viroid and plant RNA internal control

Apple scar skin viroid (ASSVd) is an important quarantine pathogen for international movement of pome germplasm as it can cause significant damage to pip fruit. A one-step real-time RT-PCR assay was developed for the rapid and sensitive detection of ASSVd. The assay was able to detect a wide range of ASSVd isolates and was highly specific compared to a published conventional RT-PCR. The detection limit of the new assay was estimated to be about 100 copies of the ASSVd target. The assay can be run as a duplex with the nad5 internal control primers and probe to simultaneously check the PCR competency of the samples therefore reducing the risk of false negatives. It is expected that this real-time RT-PCR assay will facilitate efficient testing for ASSVd by regulatory services, and will also have a wider use for the general detection of ASSVd in a range of pip fruit.

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II.

Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP PSTVd replication in the nucleoplasm generates (-)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (-)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes.

In silico prediction and validation of potential gene targets for pospiviroid-derived small RNAs during tomato infection

Viroids are small, covalently closed, circular non-coding RNA pathogens of flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we took advantage of the detailed annotation of the Arabidopsis thaliana (Arabidopsis) genome to identify sequence homologies between putative viroid-derived small RNAs (vd-sRNAs) and coding regions in the plant genome. A pool of sequence homologies among 29 species of the Pospiviroidae family and the Arabidopsis genome was analyzed. Using this strategy we identified putative host gene targets that may be involved in symptom expression in viroid-infected plants. In this communication, we report the in silico prediction and the experimental validation of pospiviroid-derived sRNAs conserved in the lower strand of the pathogenicity domain of seven viroid species infecting tomato; those vd-sRNAs targeted for cleavage the host mRNA encoding a conserved tomato WD40-repeat protein (SolWD40-repeat; SGN_U563134). Analysis of SolWD40-repeat expression indicated that this gene is down-regulated in tomato plants infected with tomato planta macho viroid (TPMVd). Furthermore, 5′ RLM-RACE revealed that the SolWD40-repeat mRNA is cleaved at the predicted target site showing complementarity to a corresponding TPMVd-sRNA identified in silico. Our approach proved to be useful for the identification of natural host genes containing sequence homologies with segments of the Pospiviroidae genome. Using this strategy we identified a functionally conserved gene in Arabidopsis and tomato, whose expression was modified during viroid infection in the host genome; regulation of this gene expression could be guided by vd-sRNA:mRNA complementarity, suggesting that the comparison of the Arabidopsis genome to viroid sequences could lead to the identification of unexpected interactions between viroid RNAs and their host.

Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo.

While biogenesis of viroid RNAs is well-known, how they decay is restricted to data involving host RNA silencing. Here we report an alternative degradation pathway operating on potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids (family Pospiviroidae). Northern-blot hybridizations with full- and partial-length probes revealed a set of PSTVd (+) subgenomic (sg)RNAs in early-infected eggplant, some partially overlapping and reaching levels comparable to those of the genomic circular and linear forms. Part of the PSTVd (+) sgRNAs were also observed in Nicotiana benthamiana (specifically in the nuclei) and tomato, wherein they have been overlooked due to their low accumulation. Primer extensions of representative (+) sgRNAs failed to detect a common 5′ terminus, excluding that they could result from aborted transcription initiated at one specific site. Supporting this view, 5′- and 3′-RACE indicated that the (+) sgRNAs have 5′-OH and 3′-P termini most likely generated by RNase-mediated endonucleolytic cleavage of longer precursors. These approaches also unveiled PSTVd (−) sgRNAs with features similar to their (+) counterparts. Our results provide a mechanistic insight on how viroid decay may proceed in vivo during replication, and suggest that synthesis and decay of PSTVd strands might be coupled as in mRNA.