Synthesis Of Double-stranded RNA Research Articles

Effective Control of Sclerotinia Stem Rot in Canola Plants Through Application of Exogenous Hairpin RNA of Multiple Sclerotinia sclerotiorum Genes.

Sclerotinia stem rot is a globally destructive plant disease caused by Sclerotinia sclerotiorum. Current management of Sclerotinia stem rot primarily relies on chemical fungicides and crop rotation, raising environmental concerns. In this study, we developed an eco-friendly RNA bio-fungicide targeting S. sclerotiorum. Six S. sclerotiorum genes were selected for double-stranded RNA (dsRNA) synthesis. Four genes, a chitin-binding domain, mitogen-activated protein kinase, oxaloacetate acetylhydrolase, and abhydrolase-3, were combined to express hairpin RNA in Escherichia coli HT115. The effect of application of total RNA extracted fromE. coliHT115 expressing hairpin RNA on disease progressive and necrosis lesions was evaluated. Gene expression analysis using real-time PCR showed silencing of the target genes using 5 ng/µl of dsRNA in a fungal liquid culture. A detached leaf assay and greenhouse application of dsRNA on canola stem and leaves showed variation in the reduction of necrosis symptoms by dsRNA of different genes, with abhydrolase-3 being the most effective. The dsRNA from a combination of four genes reduced disease severity significantly (P = 0.01). Plants sprayed with hairpin RNA from four genes had lesions that were almost 30% smaller than those of plants treated with abhydrolase-3 alone, in lab and greenhouse assays. The results of this study highlight the potential of RNA interference to manage diseases caused by S. sclerotiorum; however, additional research is necessary to optimize its efficacy.

The Single Distinct Leader Protease Encoded by Alpinia oxyphylla Mosaic Virus (Genus Macluravirus) Suppresses RNA Silencing Through Interfering with Double-Stranded RNA Synthesis.

The genomic 5'-terminal regions of viruses in the family Potyviridae (potyvirids) encode two types of leader proteases: serine-protease (P1) and cysteine-protease (HCPro), which differ greatly in the arrangement and sequence composition among inter-genus viruses. Most potyvirids have the same tandemly arranged P1 and HCPro, whereas viruses in the genus Macluravirus encode a single distinct leader protease, a truncated version of HCPro with yet-unknown functions. We investigated the RNA silencing suppression (RSS) activity and its underpinning mechanism of the distinct HCPro from alpinia oxyphylla mosaic macluravirus (aHCPro). Sequence analysis revealed that macluraviral HCPros have obvious truncations in the N-terminal and middle regions when aligned to their counterparts in potyviruses (well-characterized viral suppressors of RNA silencing). Nearly all defined elements essential for the RSS activity of potyviral counterparts are not distinguished in macluraviral HCPros. Here, we demonstrated that aHCPro exhibits a similar anti-silencing activity with the potyviral counterpart. However, aHCPro fails to block both the local and systemic spreading of RNA silencing. In line, aHCPro interferes with the dsRNA synthesis, an upstream step in the RNA silencing pathway. Affinity-purification and NanoLC-MS/MS analysis revealed that aHCPro has no association with core components or their potential interactors involving in dsRNA synthesis from the protein layer. Instead, the ectopic expression of aHCPro significantly reduces the transcript abundance of RDR2, RDR6, SGS3, and SDE5. This study represents the first report on the anti-silencing function of Macluravirus-encoded HCPro and the underlying molecular mechanism.

Trans-kingdom expression of an insect endogenous microRNA in rice enhances resistance to striped stem borer Chilo suppressalis.

The striped stem borer (SSB), Chilo suppressalis Walker, is a major pest of rice worldwide. Breeding of transgenic rice expressing Bacillus thuringiensis (Bt) toxins is a powerful strategy to control SSB. However, pests may evolve certain resistance to Bt toxins in transgenic plants. Hence, new controlling strategies must be continuously developed. We successfully generated SSB-resistant rice (csu-53) expressing the artificial microRNA (amiRNA) of SSB endogenous miRNA (csu-novel-miR53) through the RNAi-based technology. Feeding assays demonstrated that csu-53 rice inhibited larval growth, delayed pupation time, and reduced pupal weight and eclosion rate of SSB larva. In a 10-day feeding experiment, the miRNA mimic of csu-novel-miR53 also suppressed larval growth and more importantly increased larval mortality. Transcriptome analysis identified 28 differentially expressed unigenes (DEGs) in the midgut between SSB larvae fed on csu-53 rice and the wild type. One DEG (DN90065_c0_g12) validated by qRT-PCR had a predicted target site of csu-novel-miR53. In addition, in vitro double-stranded RNA synthesis and further feeding assay proved that DN90065_c0_g12 is most likely the target of csu-novel-miR53. amiRNA-mediated strategy can be applied to the development of insect-resistant crops, and the novel amiRNA csu-novel-miR53 of SSB has important application potential in developing SSB resistant rice. © 2021 Society of Chemical Industry.

Structure of RNA-dependent RNA Polymerase 2 (RDR2) and its implications for double-stranded RNA synthesis in gene silencing

Structure of RNA-dependent RNA Polymerase 2 (RDR2) and its implications for double-stranded RNA synthesis in gene silencing

Vitellogenin regulates estrogen-related receptor expression by crosstalk with the JH and IIS-TOR signaling pathway in Polyrhachis vicina Roger (Hymenoptera, Formicidae)

The Estrogen-related receptor (ERR) can regulate the growth and development, metabolism, reproduction, and other physiological activities of insects, but its specific mechanism of action is still unclear. The aim of this study was to explore the relationship between expression of ERR and Vitellogenins (Vg) and the juvenile hormone (JH) and insulin/insulin-like growth factor/target of rapamycin (IIS/TOR) signaling pathways in Polyrhachis vicina Roger. P. vicina was used as the experimental model to clone the PvVg gene, perform double-stranded RNA synthesis and delivery and observe the effects of pharmacological treatments. The full-length PvVg cDNA product is 5586 bp. Higher PvVg mRNA expression was seen in the pupa and adults, and varying levels were seen in the different body parts of three different castes. RNA interference of PvVg expression led to disturbed development, an abnormal phenotype, and high mortality. PvVg RNAi also led to a reduction in mRNA levels of PvERR, ultraspiracle (PvUSP), forkhead box protein O (PvFOXO) and PvTOR genes in fourth instar larval, but a significant increase was seen in pupa and females. No significant change was seen in workers and males. After PvVg knockdown, application of exogenous JHIII reduced the expression of these genes in pupa and females, increased expression in workers, and decreased PvUSP mRNA expression in males. Both protein and mRNA expression levels of PvFOXO were affected by PvVg RNAi. PvERR RNAi increased PvVg expression in pupa and females and Kruppel-homolog 1 (PvKr-h1) and PvFOXO expression in males. The results of this study suggest that there is an interaction between PvERR and PvVg, and that crosstalk with the JH and IIS/TOR signaling pathways can affect development and reproduction. This effect is caste and developmental stage specific. We also speculate that the FOXO/USP complex participates in JH regulation of PvVg in P. vicina.

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points.

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites.

The rotavirus polymerase VP1 mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein VP2. Transcription (positive-strand RNA [+RNA] synthesis) by VP1 occurs within double-layered particles (DLPs), while genome replication (double-stranded RNA [dsRNA] synthesis) by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet, the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral 5-fold axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264 to 267, 547 to 550, 614 to 620, 968 to 980, and 1022 to 1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 with regard to polymerase activity. We engineered 19 recombinant VP1 (rVP1) proteins that contained single- or multipoint alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their nonmutated contact sites. These results move us toward a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation.IMPORTANCE Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase VP1 mediates all stages of viral RNA synthesis, and it requires the core shell protein VP2 for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance the understanding of a key step of the rotavirus replication cycle.

RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene.

The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100–200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways.

Viral RNA at Two Stages of Reovirus Infection Is Required for the Induction of Necroptosis

Necroptosis, a regulated form of necrotic cell death, requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I interferon (IFN). While these events that occur prior to the de novo synthesis of viral RNA are required for the induction of necroptosis, they are not sufficient. The induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of double-stranded RNA (dsRNA) within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus.IMPORTANCE An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is understood for only a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis.

Cellular Casein Kinase 2 and Protein Phosphatase 2A Modulate Replication Site Assembly of Bluetongue Virus

A number of cytoplasmic replicating viruses produce cytoplasmic inclusion bodies or protein aggregates; however, a hallmark of viruses of the Reoviridae family is that they utilize these sites for purposes of replication and capsid assembly, functioning as viral assembly factories. Here we have used bluetongue virus (BTV) as a model system for this broad family of important viruses to understand the mechanisms regulating inclusion body assembly. Newly synthesized viral proteins interact with sequestered viral RNA molecules prior to capsid assembly and double-stranded RNA synthesis within viral inclusion bodies (VIBs). VIBs are predominantly comprised of a BTV-encoded non-structural protein 2 (NS2). Previous in vitro studies indicated that casein kinase 2 (CK2) mediated the phosphorylation of NS2, which regulated the propensity of NS2 to form larger aggregates. Using targeted pharmacological reagents, specific mutation in the viral genome by reverse genetics and confocal microscopy, here we demonstrate that CK2 activity is important for BTV replication. Furthermore, we show that a novel host cell factor, protein phosphatase 2A, is involved in NS2 dephosphorylation and that, together with CK2, it regulates VIB morphology and virus replication. Thus, these two host enzymes influence the dynamic nature of VIB assembly/disassembly, and these concerted activities may be relevant to the assembly and the release of these cores from VIBs.

Poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsRNA and the induction of innate antiviral responses.

Poxvirus replication involves synthesis of double-stranded RNA (dsRNA), which can trigger antiviral responses by inducing phosphorylation-mediated activation of protein kinase R (PKR) and stimulating 2'5'-oligoadenylate synthetase (OAS). PKR inactivates the translation initiation factor eIF2α via phosphorylation, while OAS induces the endonuclease RNase L to degrade RNA. We show that poxvirus decapping enzymes D9 and D10, which remove caps from mRNAs, inhibit these antiviral responses by preventing dsRNA accumulation. Catalytic site mutations of D9 and D10, but not of either enzyme alone, halt vaccinia virus late protein synthesis and inhibit virus replication. Infection with the D9-D10 mutant was accompanied by massive mRNA reduction, cleavage of ribosomal RNA, and phosphorylation of PKR and eIF2α that correlated with a ∼ 15-fold increase in dsRNA compared to wild-type virus. Additionally, mouse studies show extreme attenuation of the mutant virus. Thus, vaccinia virus decapping, in addition to targeting mRNAs for degradation, prevents dsRNA accumulation and anti-viral responses.

Intronic regions of plant genes potentially encode RDR (RNA-dependent RNA polymerase)-dependent small RNAs.

Recent research has linked the non-coding intronic regions of plant genes to the production of small RNAs (sRNAs). Certain introns, called 'mirtrons' and 'sirtrons', could serve as the single-stranded RNA precursors for the generation of microRNA and small interfering RNA, respectively. However, whether the intronic regions could serve as the template for double-stranded RNA synthesis and then for sRNA biogenesis through an RDR (RNA-dependent RNA polymerase)-dependent pathway remains unclear. In this study, a genome-wide search was made for the RDR-dependent sRNA loci within the intronic regions of the Arabidopsis genes. Hundreds of intronic regions encoding three or more RDR-dependent sRNAs were found to be covered by dsRNA-seq (double-stranded RNA sequencing) reads, indicating that the intron-derived sRNAs were indeed generated from long double-stranded RNA precursors. More interestingly, phase-distributed sRNAs were discovered on some of the dsRNA-seq read-covered intronic regions, and those sRNAs were largely 24 nt in length. Based on these results, the opinion is put forward that the intronic regions might serve as the genomic origins for the RDR-dependent sRNAs. This opinion might add a novel layer to the current biogenesis model of the intron-derived sRNAs.

An effective and convenient method for the delivery of apple latent spherical virus (ALSV)-based vectors into plant cells by agroinoculation.

Virus infection leads to the synthesis of double-stranded RNA during virus replication, and then this infection produces small RNA molecules in the antiviral RNA silencing pathway. Here, we develop an Agrobacterium-mediated inoculation system for ALSV-based vectors. This system is more effective and convenient for inoculation of the ALSV vectors into plants compared to direct inoculation of ALSV-RNA2-based vectors in pUC plasmids reported previously. In addition, cointroduction of various plant viral RNA-silencing suppressors increased the efficiency of agroinoculation of the ALSV-based vector. An ALSV vector could be successfully used to silence an endogenous gene in plants. Therefore, the ALSV-based VIGS/agroinoculation system provides a valuable tool for functional genomics among a broad range of plant species.

RNA silencing is induced by the expression of foreign recombinant products in transgenic rice

RNA silencing plays important roles in the regulation of gene expression in eukaryotes. We previously reported that RNA silencing of a linked endogenous gene and a transgene in transgenic rice seeds can be induced by the expression of foreign recombinant mGLP-1, which acts as a silencing-inducible sequence through RNA interference. In this study, we found that the induction of RNA silencing by foreign transgenes is not restricted to mGLP-1 but is observed in many other genes as a relatively general phenomenon, as several foreign genes were involved in inducing RNA silencing in the same manner as mGLP-1 in transgenic rice. We detected 21–24-nt siRNAs using both sense and antisense probes specific to the silenced genes in both the leaves and endosperm of transgenic rice plants. Moreover, read-through transcripts were consistently observed in silenced transgenic rice plants. Taken together, these results suggest that proper transcription termination was prevented in these plants, and the highly divergent 3′-end transcripts served as templates for double-stranded RNA synthesis, resulting in the degradation of the target genes via siRNA.

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor.

RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.

Development of Continuous Cell Culture of Brown Planthopper To Trace the Early Infection Process of Oryzaviruses in Insect Vector Cells

Rice ragged stunt virus (RRSV), an oryzavirus in the family Reoviridae, is transmitted by the brown planthopper, Nilaparvata lugens, in a persistent-propagative manner. Here, we established a continuous cell line of brown planthopper to investigate the mechanism underlying the formation of the viroplasm, the putative site for viral replication and assembly, during infection of RRSV in its insect vector cells. Within 24 h of viral infection of cultured cells, the viroplasm had formed and contained the viral nonstructural proteins Pns6 and Pns10, known to be constituents of viroplasm. Core capsid protein P3, core particles, and newly synthesized viral RNAs were accumulated inside the viroplasm, while outer capsid protein P8 and virions were accumulated at the periphery of the viroplasm, confirming that the viroplasm induced by RRSV infection was the site for viral replication and assembly. Pns10 formed viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm matrix was largely composed of Pns10. Pns6 was recruited in the viroplasm by direct interaction with Pns10. Core capsid protein P3 was recruited to the viroplasm through specific association with Pns6. Knockdown of Pns6 and Pns10 expression using RNA interference inhibited viroplasm formation, virion assembly, viral protein expression, and viral double-stranded RNA synthesis. Thus, the present study shows that both Pns6 and Pns10 of RRSV play important roles in the early stages of viral life cycle in its insect vector cells, by recruiting or retaining components necessary for viral replication and assembly. The brown planthopper, a commonly distributed pest of rice in Asia, is the host of numerous insect endosymbionts, and the major vector of two rice viruses (RRSV and rice grassy stunt virus). For the first time, we successfully established the continuous cell line of brown planthopper. The unique uniformity of brown planthopper cells in the monolayer can support a consistent, synchronous infection by endosymbionts or viral pathogens, improving our understanding of molecular insect-microbe interactions.

A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 “lead-like” compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC50 of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

High-throughput purification of double-stranded RNA molecules using convective interaction media monolithic anion exchange columns

Recent advances in the field of RNA interference and new cost-effective approaches for large-scale double-stranded RNA (dsRNA) synthesis have fuelled the demand for robust high-performance purification techniques suitable for dsRNA molecules of various lengths. To address this issue, we developed an improved dsRNA purification method based on anion exchange chromatography utilizing convective interaction media (CIM) monolithic columns. To evaluate column performance we synthesized a selection of dsRNA molecules (58–1810bp) in a one-step enzymatic reaction involving bacteriophage T7 DNA-dependent RNA polymerase and phi6 RNA-dependent RNA polymerase. In addition, small interfering RNAs (siRNAs) of 25–27bp were generated by Dicer digestion of the genomic dsRNA of bacteriophage phi6. We demonstrated that linearly scalable CIM monolithic quaternary amine (QA) columns can be used as a fast and superior alternative to standard purification methods (e.g. LiCl precipitation) to obtain highly pure dsRNA preparations. The impurities following Dicer treatment were quickly and efficiently removed with the QA CIM monolithic column, yielding siRNA molecules of high purity suitable for potential therapeutic applications. Moreover, baseline separation of dsRNA molecules up to 1kb in non-denaturing conditions was achieved.

RNA Silencing Induced by an Artificial Sequence That Prevents Proper Transcription Termination in Rice

Posttranscriptional gene silencing (PTGS) is a sequence-specific mRNA degradation caused by small RNA, such as microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are generated from MIRNA loci, whereas siRNAs originate from various sources of double-stranded RNA. In this study, an artificial RNA silencing inducible sequence (RSIS) was identified in rice (Oryza sativa). This sequence causes PTGS of 5' or 3' flanking-sequence-containing genes. Interestingly, two target genes can be simultaneously suppressed by linking a unique target sequence to either the 5' or 3' end of RSIS. Multiple gene suppression can be also achieved though a single transformation event by incorporating the multisite gateway system. Moreover, RSIS-mediated PTGS occurs in nuclei. Deep sequencing of small RNAs reveals that siRNAs are produced from RSIS-expressing cassettes and transitive siRNAs are produced from endogenous target genes. Furthermore, siRNAs are typically generated from untranscribed transgene terminator regions. The read-through transcripts from the RSIS-expression cassette were consistently observed, and most of these sequences were not polyadenylated. Collectively, this data indicates that RSIS inhibits proper transcription termination. The resulting transcripts are not polyadenylated. These transcripts containing RSIS become templates for double-stranded RNA synthesis in nuclei. This is followed by siRNA production and target degradation of target genes.

Interaction of Calpactin Light Chain (S100A10/p11) and a Viral NS Protein Is Essential for Intracellular Trafficking of Nonenveloped Bluetongue Virus

Bluetongue virus (BTV), a member of the Reoviridae family, is an insect-borne animal pathogen. Virus release from infected cells is predominantly by cell lysis, but some BTV particles are also released from the plasma membrane. The nonstructural protein NS3 has been implicated in this process. Using alternate initiator methionine residues, NS3 is expressed as a full-length protein and as a truncated variant that lacks the initial 13 residues, which, by yeast-two hybrid analyses, have been shown to interact with a cellular trafficking protein S100A10/p11. To understand the physiological significance of this interaction in virus-infected cells, we have used reverse genetics to investigate the roles of NS3 and NS3A in virus replication and localization in both mammalian and insect vector-derived cells. A virus expressing NS3 but not NS3A was able to propagate in and release from mammalian cells efficiently. However, growth of a mutant virus expressing only NS3A was severely attenuated, although protein expression, replication, double-stranded RNA (dsRNA) synthesis, and particle assembly in the cytoplasm were observed. Two of three single-amino-acid substitutions in the N-terminal 13 residues of NS3 showed phenotypically similar effects. Pulldown assay and confocal microscopy demonstrated a lack of interaction between NS3 and S100A10/p11 in mutants with poor replication. The role of NS3/NS3A was also assessed in insect cells where virus grew, albeit with a reduced titer. Notably, however, while wild-type particles were found within cytoplasmic vesicles in insect cells, mutant viruses were scattered throughout the cytoplasm and not confined to vesicles. These results provide support for a role for the extreme amino terminus of NS3 in the late stages of virus growth in mammalian cells, plausibly in egress. However, both NS3 and NS3A were required for efficient BTV growth in insect cells.