Length, mass, and denaturation of double-stranded RNA molecules compared with DNA

The contour lengths of linear, double-stranded (ds) RNAs from mycovirus PcV and Pseudomonas bacteriophage phi 6 have been measured with samples prepared for the electron microscope from 0.05 to 0.5 M NH4Cl solutions. A linear dependence of contour length on the logarithm of ionic strength was found and compared with that of dsDNA (pBR322, linearized and open-circular forms). Conditions for molecular weight determinations of any natural dsRNA by electron microscopy have been established, and the method has been calibrated with phi 6 dsRNA of known nucleotide sequence. The results imply that dsRNA in 0.20 M NH4Cl solution has a rise per basepair of 0.271 nm, which is shorter than that in the A-conformation (4%) and in the A'-conformation (10%). The thermal behavior of dsRNA in terms of melting temperature and exhibition of fine structure of melting curves was found to be generally similar to that of dsDNA, as expected from the literature. Folding of dsRNA in ethanolic solution was similar to that of dsDNA. However, in contrast to dsDNA, coiled coils could not be induced by ethanol, which is consistent with dsRNA being stiffer than dsDNA. Concerning dsDNA, the results show that a contraction in rise per basepair by 0.1 nm is coupled with an increase in the winding angle between basepairs by 0.47 degrees, as qualitatively predicted by polyelectrolyte theory.     

Resistance of bacterial protein synthesis to double-stranded RNA

Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.     

Complex of fd gene 5 protein and double-stranded RNA

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.     

A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust

Application of RNA interference (RNAi) for insect pest management is limited by variable efficiency of RNAi in different insect species. In Locusta migratoria, RNAi is highly efficient through injection of dsRNA, but oral delivery of dsRNA is much less effective. Efforts to understand this phenomenon have shown that dsRNA is more rapidly degraded in midgut fluid than in hemolymph due to nuclease enzyme activity. In the present study, we identified and characterized two full-length cDNAs of double-stranded RNA degrading enzymes (dsRNase) from midgut of L. migratoria, which were named LmdsRNase2 and LmdsRNase3. Gene expression analysis revealed that LmdsRNase2 and LmdsRNase3 were predominantly expressed in the midgut, relatively lower expression in gastric caeca, and trace expression in other tested tissues. Incubation of dsRNA in midgut fluid from LmdsRNase3-suppressed larvae or control larvae injected with dsGFP resulted in high levels of degradation; however, dsRNA incubated in midgut fluid from LmdsRNase2-suppressed larvae was more stable, indicating LmdsRNase2 is responsible for dsRNA degradation in the midgut. To verify the biological function of LmdsRNase2 in vivo, nymphs were injected with dsGFP, dsLmdsRNase2 or dsLmdsRNase3 and chitinase 10 (LmCht10) or chitin synthase 1 (LmCHS1) dsRNA were orally delivered. Mortality associated with reporter gene knockdown was observed only in locusts injected with dsLmdsRNase2 (48% and 22%, for dsLmCht10 and dsLmCHS1, respectively), implicating LmdsRNase2 in reducing RNAi efficiency. Furthermore, recombinantly expressed LmdsRNase2 fusion proteins degraded dsRNA rapidly, whereas LmdsRNase3 did not. These results suggest that rapid degradation of dsRNA by dsRNase2 in the midgut is an important factor causing low RNAi efficiency when dsRNA is orally delivered in the locust.     

Recognition of double-stranded RNA by proteins and small molecules

Molecular recognition of double-stranded RNA (dsRNA) is a key event for numerous biological pathways including the trafficking, editing, and maturation of cellular RNA, the interferon antiviral response, and RNA interference. Over the past several years, our laboratory has studied proteins and small molecules that bind dsRNA with the goal of understanding and controlling the binding selectivity. In this review, we discuss members of the dsRBM class of proteins that bind dsRNA. The dsRBM is an approximately 70 amino acid sequence motif found in a variety of dsRNA-binding proteins. Recent results have led to a new appreciation of the ability of these proteins to bind selectivity to certain sites on dsRNA. This property is discussed in light of the RNA selectivity observed in the function of two proteins that contain dsRBMs, the RNA-dependent protein kinase (PKR) and an adenosine deaminase that acts on dsRNA (ADAR2). In addition, we introduce peptide-acridine conjugates (PACs), small molecules designed to control dsRBM-RNA interactions. These intercalating molecules bear variable peptide appendages at opposite edges of an acridine heterocycle. This design imparts the potential to exploit differences in groove characteristics and/or base-pair dynamics at binding sites to achieve selective binding.     

Visualization of DNA and RNA molecules, and protein-DNA complexes for electron microscopy

This article provides step-by step instructions for the preparation of double- and single-stranded DNA and RNA molecules and protein-DNA complexes for electron microscopy (EM). Absorption, spreading, staining, dark-field imaging, and metal shadowing techniques are described in detail. A number of examples are illustrated on analysis of DNA replication, DNA repair and DNA recombination to demonstrate the usefulness of the technique for EM visualisation. Application of immunogold labeling of specific protein in DNA-protein complexes is also covered.     

Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4

Bacteriophage N4 encapsidates a 3500-aa-long DNA-dependent RNA polymerase (vRNAP), which is injected into the host along with the N4 genome upon infection. The three-dimensional structures of wild-type and mutant N4 viruses lacking gp17, gp50, or gp65 were determined by cryoelectron microscopy. The virion has an icosahedral capsid with T = 9 quasi-symmetry that encapsidates well-organized double-stranded DNA and vRNAP. The tail, attached at a unique pentameric vertex of the head, consists of a neck, 12 appendages, and six ribbons that constitute a non-contractile sheath around a central tail tube. Comparison of wild-type and mutant virus structures in conjunction with bioinformatics established the identity and virion locations of the major capsid protein (gp56), a decorating protein (gp17), the vRNAP (gp50), the tail sheath (gp65), the appendages (gp66), and the portal protein (gp59). The N4 virion organization provides insight into its assembly and suggests a mechanism for genome and vRNAP transport strategies utilized by this unique system.     

Cloning and molecular analysis of double-stranded RNA associated with grapevine leafroll disease*

Eight major dsRNA species ranging from 1.0 to 19.5 kbp were detected in a low-yielding clone of Sultana (Thompson seedless) grape (Vitis vinifera L., cv. Sultana, clone B4L) affected leafroll disease. Using total dsRNA from this Sultana line as template, a number of cDNA clones were produced. The clones were used as probes for northern blot analysis of dsRNA extracted from Sultana B4L, and from six other grapevine leafroll-infected Sultana sources differing in yield performance. Based on the hybridisation of each probe with dsRNA bands from various Sultanas, the cDNA clones could be divided into three groups. One group of cDNA clones hybridised to high molccular weight dsRNA (19.5 kbp) from two low-yielding Sultanas, another group hybridised to high M_r dsRNA from three low-yielding Sultanas and the third group hybridised to a number of smaller dsRNA species ranging in size between 1.15 and 6.5 kbp. Using the latter cDNA clones, the sequence of 965 nucleotides at the 5′-end of a 1.15 kbp dsRNA (dsRNA 6) of B4L Sultana was determined. This RNA contains an open reading frame encoding a putative protein of M, = 33 441 with no homology to known protein sequences. The sequence of dsRNA 6 was found to overlap larger dsRNAs of sizes between 2.2 to 6.5 kbp. This allowed us to determine the sequence upstream of the 5′-end of the positive strand of dsRNA 6. The nucleotide sequence neighbouring the 5′-end of the positive strand of dsRNA 6 conforms to a consensus sequence proposed as a subgenomic promoter element for the coat protein gene of positive strand RNA plant viruses. The results indicate that more than one virus was present in Sultana B4L and that dsRNA 6 may be a subgenomic species of viral origin.     

dsRNA with 5′ overhangs contributes to endogenous and antiviral RNA silencing pathways in plants

In plants, SGS3 and RNA‐dependent RNA polymerase 6 (RDR6) are required to convert single‐ to double‐stranded RNA (dsRNA) in the innate RNAi‐based antiviral response and to produce both exogenous and endogenous short‐interfering RNAs. Although a role for RDR6‐catalysed RNA‐dependent RNA polymerisation in these processes seems clear, the function of SGS3 is unknown. Here, we show that SGS3 is a dsRNA‐binding protein with unexpected substrate selectivity favouring 5′‐overhang‐containing dsRNA. The conserved XS and coiled‐coil domains are responsible for RNA‐binding activity. Furthermore, we find that the V2 protein from tomato yellow leaf curl virus, which suppresses the RNAi‐based host immune response, is a dsRNA‐binding protein with similar specificity to SGS3. In competition‐binding experiments, V2 outcompetes SGS3 for substrate dsRNA recognition, whereas a V2 point mutant lacking the suppressor function in vivo cannot efficiently overcome SGS3 binding. These findings suggest that SGS3 recognition of dsRNA containing a 5′ overhang is required for subsequent steps in RNA‐mediated gene silencing in plants, and that V2 functions as a viral suppressor by preventing SGS3 from accessing substrate RNAs.     

Association of particles that contain double-stranded RNAs with algal chloroplasts and mitochondria

Linear dsRNAs (double-stranded RNAs) belonging to several distinct size classes were found to be localized in chloroplasts and mitochondria of Bryopsis spp., raising the possibility that these dsRNAs are prokaryotic in nature. The algal cytosol and nuclei did not contain dsRNAs. The amount of the dsRNAs in the organelles appeared constant, and there were about 500 copies per chloroplast. The four major dsRNAs from Bryopsis chloroplasts were about 2 kbp (kilobase pairs) in length and originated from discrete isometric particles of about 25 nm diameter. These virus-like particles were purified by CsCl density gradient centrifugation after extraction from isolated chloroplasts with chloroformbutanol and subsequent precipitation with polyethylene glycol. They had a buoyant density of about 1.40 g · cm^–3 and contained four major and three minor proteins. Mitochondrial dsRNAs were about 4.5 kbp in length and formed less-stable particles of about 40 nm in diameter with a buoyant density of 1.47 g · cm^–3. Some observations support the hypothesis that vertical transmission of the protein-coated, non-infectious dsRNAs occurs within cell organelles. Double-stranded RNAs of various sizes were found in most green, red, and brown algae. The characteristics of the algal dsRNAs are compared with those of dsRNAs from higher plants and the biological significance of the dsRNAs in cell organelles is discussed.Abbreviations dsRNA double-stranded RNA - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl 0.015 M sodium citrate - PAGE polyacrylamide gel electrophoresis The authors would like to express their gratitude to Dr. T. Natsuaki, Utsunomiya University, and Dr. D. Hosokawa, Tokyo University of Agriculture and Technology, for their helpful suggestions throughout this research. They are also much indebted to Dr. B. Wang, Institute of Genetics, Academia Sinica, Beijing, PRC, for his suggestions on rice dsRNA, and to Dr. T. Kohbara, Senshu University, on Bryopsis cells. Sincere thanks are also due to Dr. T. Misonou, Yamanashi University, and Dr. K. Masuda, Akita Prefectural College of Agriculture, for supplying plant materials; to Dr. N. Sonoki, Azabu University, for nucleotide analysis of dsRNAs; and to Y. Koshino for technical assistance. This research was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.     

Solution structures of the double-stranded RNA-binding domains from RNA helicase A

RNA helicase A (RHA) is a highly conserved protein with multifaceted functions in the gene expression of cellular and viral mRNAs. RHA recognizes highly structured nucleotides and catalytically rearranges the various interactions between RNA, DNA, and protein molecules to provide a platform for the ribonucleoprotein complex. We present the first solution structures of the double-stranded RNA-binding domains (dsRBDs), dsRBD1 and dsRBD2, from mouse RHA. We discuss the binding mode of the dsRBDs of RHA, in comparison with the known dsRBD structures in their complexes. Our structural data provide important information for the elucidation of the molecular reassembly mediated by RHA.     

Presence and distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix

Double-stranded (ds)RNA of various types was detected in 65 (21.8%) of 298 isolates from vegetative hyphae of Rosellinia necatrix by electrophoresis, but dsRNA was not detected from 39 ascosporic isolates. There were 45 distinct dsRNA profiles in the 65 isolates: they varied in the number of electrophoretic bands from 1 to 12 and in size from less than 1000 bp to more than 10 kbp. Each dsRNA profile was unique to each locality. dsRNAs having the same profiles were restricted to isolates of the same mycelial compatibility groups (MCG) from the same trees, with an exception where different profiles were detected in different isolates of the same MCGs. Received: May 7, 2001 / Accepted: September 5, 2001     

A complex between met-tRNA F and native 40S subunits in reticulocyte lysates and its disappearance during incubation with double-stranded RNA

We describe the detection of a complex between met tRNA_F and native 40S ribosomal subunits in the crude reticulocyte lysate which synthesises globin at a rate close to that of the intact cells. We show that double-stranded RNA, a highly specific and powerful inhibitor of initiation in this system, causes the complex to disappear. This not only casts light on the mechanism of action of double-stranded RNA as an inhibitor of protein synthesis, but also suggests that the complex between 40S subunits and met tRNA_F, which other authors have noted using highly fractionated systems, does indeed play an integral role in the initiation of protein synthesis in eukaryotic cells.