Junctophilin-2 is a double-stranded RNA-binding protein that regulates cardiomyocyte-autonomous innate immune response.

Junctophilin-2 (JPH2) is traditionally recognized as a cardiomyocyte-enriched structural protein that anchors the junction between the plasma membrane and the endo/sarcoplasmic reticulum, facilitating excitation-induced cardiac contraction. In this study, we uncover a novel function of JPH2 as a double-stranded RNA (dsRNA)-binding protein, which forms complexes with dsRNA both in vitro and in cells. Stimulation by cytosolic dsRNA enhances the interaction of JPH2 with the dsRNA sensor MDA5. Notably, JPH2 inhibits MDA5's binding to its dsRNA ligand, likely by sequestering the dsRNA. Silencing JPH2 in cardiomyocytes increased the interaction between MDA5 and its dsRNA ligands, activated the MAVS/TBK1 signaling, and triggered spontaneous interferon-beta (IFNb1) production in the absence of foreign pathogen. Mouse hearts deficient in JPH2 exhibited upregulation of innate immune signaling cascade. Collectively, these findings identify JPH2 as a regulator of dsRNA sensing and highlight its role in suppressing the automatic activation of innate immune responses in cardiomyocytes, suggesting the cytosolic surface of the endo/sarcoplasmic reticulum as a hub for dsRNA sequestration.

Cationic Polystyrene Latex Nanocarriers for Immunostimulatory Long Double-Stranded RNA Delivery to Ovarian Cancer Cells.

Long double-stranded (ds)RNA, a potent stimulator of type I interferon and the innate immune response. In the present study, we demonstrated, for the first time, the efficacy of cationic polystyrene latex nanostructures (clNPs) as a dsRNA carrier, improving cellular delivery and robustly potentiating the immunostimulatory capacity of dsRNA in the ovarian cancer cell line SKOV3. The clNPs complexed with an in vitro transcribed dsRNA molecule, were bound by SKOV3 cells, and had increased cellular association compared to uncomplexed clNPs. clNPs complexed with dsRNA induced a more robust innate immune response compared to dsRNA alone. Transcript expression of two interferon-stimulated genes, were increased 47- and 108-fold over dsRNA and induced a significant antiviral state against vesicular-stomatitis virus, resulting in a 3.3-fold improvement on the efficacy of dsRNA. These data highlight the potential of polystyrene latex nanostructures as dsRNA carriers for anticancer immunotherapies, improving the uptake and efficacy of the nucleic acid.

Simultaneous Silencing of Gut Nucleases and a Vital Target Gene by Adult dsRNA Feeding Enhances RNAi Efficiency and Mortality in Ceratitis capitata.

Ceratitis capitata, known as the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on pollinators. A more sustainable and species-specific alternative strategy may be based on double-stranded RNA (dsRNA) delivery through feeding to disrupt essential functions in pest insects, which is poorly reported in dipteran species. Previous reports in Orthoptera and Coleoptera species suggested that dsRNA degradation by specific nucleases in the intestinal lumen is among the major obstacles to feeding-mediated RNAi in insects. In our study, we experimented with three-day adult feeding using a combination of dsRNA molecules that target the expression of the ATPase vital gene and two intestinal dsRNA nucleases. These dsRNA molecules were recently tested separately in two Tephritidae species, showing limited effectiveness. In contrast, by simultaneously feeding dsRNA against the CcVha68-1, CcdsRNase1, and CcdsRNase2 genes, we observed 79% mortality over seven days, which was associated with a decrease in mRNA levels of the three targeted genes. As expected, we also observed a reduction in dsRNA degradation following RNAi against nucleases. This research illustrates the potential of utilizing molecules as pesticides to achieve mortality rates in Medfly adults by targeting crucial genes and intestinal nucleases. Furthermore, it underscores the importance of exploring RNAi-based approaches for pest management.

Differential Drug Susceptibility across Trichomonasvirus Species Allows for Generation of Varied Isogenic Clones of Trichomonas vaginalis.

Trichomonas vaginalis (Tvag) is a sexually transmitted human pathogen that is commonly infected with strains of one or more of five known species of Trichomonas vaginalis viruses (TVVs), members of genus Trichomonasvirus. TVVs are thought not to have an extracellular phase to their lifecycle and instead to be transmitted vertically from mother to daughter cells. As a result, generation of isogenic virus-positive and virus-negative sets of Tvag clones has been a major barrier to studying interactions between TVVs and their host. Nucleoside analog 2'-C-methylcytidine (2CMC) has been recently reported to clear trichomonads of infections with TVV1, TVV2, and TVV3. We used 2CMC to treat a panel of Tvag isolates that collectively harbor at least one representative strain of each TVV species and thereby provided evidence that infections with TVV4 and TVV5 can also be cleared by 2CMC. Furthermore, our results suggest a newly identified difference in drug susceptibility between TVV species. We took advantage of these susceptibility difference to generate isogenic sets of Tvag clones harboring different combinations of the five TVV species. These results provide both new insight into differences between these species and new avenues for generating tools to study the potential roles of TVVs in Tvag biology.

Development of Biolayer Interferometry (BLI)-Based Double-Stranded RNA Detection Method with Application in mRNA-Based Therapeutics and Vaccines.

Background: In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and quantitation methods, such as gel electrophoresis, ELISA, or homogeneous time-resolved fluorescence (HTRF), have low sensitivity or are time-consuming. A recently published lateral flow immunoassay (LFSA) was shown to be fast, but it lacks the sensitivity for dsRNA with uridine modifications. Methods: In this study, we provided a possible explanation for the reduced sensitivity of existing quantitation methods for dsRNA with modified uridines by characterizing the binding affinities of commonly used anti-dsRNA antibodies. Then, a rapid and sensitive biolayer interferometry (BLI) dsRNA detection assay utilizing Flock House Virus (FHV) B2 protein was developed to overcome the challenges in dsRNA detection and the reduced sensitivity. Results: This assay allows the detection of dsRNA with different uridine modifications (ψ, m1ψ, 5 moU) with similar sensitivity as dsRNA without modification. Furthermore, we demonstrated this method can be used to quantify both short and long dsRNA, as well as hairpin-structured dsRNA, providing a more comprehensive detection for dsRNA impurities. Moreover, we applied this assay to monitor dsRNA removal through a purification process. Conclusions: Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA.

Nanoparticle LDH enhances RNAi efficiency of dsRNA in piercing-sucking pests by promoting dsRNA stability and transport in plants.

Piercing-sucking pests are the most notorious group of pests for global agriculture. RNAi-mediated crop protection by foliar application is a promising approach in field trials. However, the effect of this approach on piercing-sucking pests is far from satisfactory due to the limited uptake and transport of double strand RNA (dsRNA) in plants. Therefore, there is an urgent need for more feasible and biocompatible dsRNA delivery approaches to better control piercing-sucking pests. Here, we report that foliar application of layered double hydroxide (LDH)-loaded dsRNA can effectively disrupt Panonychus citri at multiple developmental stages. MgAl-LDH-dsRNA targeting Chitinase (Chit) gene significantly promoted the RNAi efficiency and then increased the mortality of P. citri nymphs by enhancing dsRNA stability in gut, promoting the adhesion of dsRNA onto leaf surface, facilitating dsRNA internalization into leaf cells, and delivering dsRNA from the stem to the leaf via the vascular system of pomelo plants. Finally, this delivery pathway based on other metal elements such as iron (MgFe-LDH) was also found to significantly improve the protection against P. citri and the nymphs or larvae of Diaphorina citri and Aphis gossypii, two other important piercing-sucking hemipeteran pests, indicating the universality of nanoparticles LDH in promoting the RNAi efficiency and mortality of piercing-sucking pests. Collectively, this study provides insights into the synergistic mechanism for nano-dsRNA systemic translocation in plants, and proposes a potential eco-friendly control strategy for piercing-sucking pests.

Regulation of Anthocyanin Accumulation in Tomato Solanum lycopersicum L. by Exogenous Synthetic dsRNA Targeting Different Regions of SlTRY Gene.

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis.

IBR1, a novel endogenous IFIH1-binding dsRNA, governs IFIH1 activation and M1 macrophage polarisation in ARDS.

BACKGROUND: Uncontrolled inflammation caused by macrophages and monocytes plays a crucial role in worsening acute respiratory distress syndrome (ARDS). Previous studies have highlighted the importance of IFIH1 in regulating macrophage polarisation in ARDS triggered by pneumonia. However, the mechanisms by which IFIH1 is activated in ARDS remain unclear. METHODS: In this study, we utilised multiomics sequencing and molecular interaction experiments to explore the molecular mechanisms underlying IFIH1 activation in ARDS. Through the use of conditional gene knockout mice and primary cells, we demonstrated the significant role of these mechanisms in the development of ARDS. Additionally, we validated the associations between these mechanisms and ARDS by quantitative PCR analysis of CD14+ cells obtained from the peripheral blood of 140 ARDS patients. RESULTS: Our investigation revealed that lipopolysaccharide, a critical component derived from Gram-negative bacteria, activated IFIH1 by upregulating a novel transcript known as IFIH1-binding RNA1 (IBR1) in monocytes and macrophages. Specifically, as an endogenous double-stranded RNA, IBR1 bind to the helicase domain of IFIH1 because of its unique double-stranded structure. Deletion of IBR1 significantly reduced the activation of IFIH1, M1 polarisation of macrophages, and inflammatory lung injury in ARDS. Moreover, IBR1 directly induced M1 polarisation of macrophages and ARDS, whereas deletion of IFIH1 inhibited IBR1-induced macrophage M1 polarisation and inflammatory lung injury. Importantly, we observed a notable increase in IBR1 expression in ARDS patients with pneumonia caused by Gram-negative bacteria. Furthermore, we demonstrated that the delivery of IFIH1 mutants through exosomes effectively counteracted IBR1, thereby reducing pulmonary inflammation and alleviating lung injury. CONCLUSIONS: This study revealed a novel mechanism involving IBR1, an endogenous double-stranded RNA (dsRNA) that binds to IFIH1, shedding light on the complex process of macrophage polarisation in ARDS. The administration of IFIH1 variants has the potential to eliminate pulmonary dsRNA and alleviate inflammatory lung injury in ARDS. HIGHLIGHTS: In monocytes and macrophages, the endogenous double-stranded RNA, IFIH1-binding RNA 1 (IBR1), binds to the helicase domain of IFIH1 because of its unique double-stranded structure. IBR1 plays a significant role in macrophage polarisation and the development of acute respiratory distress syndrome (ARDS) induced by Gram-negative bacteria or lipopolysaccharide (LPS). Administration of IFIH1 variants has potential for eliminating pulmonary IBR1 and reducing inflammatory lung injury in ARDS patients.

A novel role of ETV6 as a pro-viral factor in host response by inhibiting TBK1 phosphorylation.

E26-transforming specific (ETS) variant 6 (ETV6) is a transcription factor regulating the expression of interferon stimulating genes (ISGs) and involved in the embryonic development and hematopoietic regulation, but the role of ETV6 in host response to virus infection is not clear. In this study, we show that ETV6 was upregulated in DF-1 cells with poly(I:C) stimulation or IBDV, AIV and ARV infection via engagement of dsRNA by MDA5. Overexpression of ETV6 in DF-1 cells markedly inhibited IBDV-induced type I interferon (IFN-I) and ISGs expressions. In contrast, knockdown, or knockout of ETV6 remarkably inhibited IBDV replication via promoting IFN-I response. Furthermore, our data show that ETV6 negatively regulated host antiviral response to IBDV infection by interaction with TANK binding kinase 1 (TBK1) and subsequently inhibited its phosphorylation. These results uncovered a novel role of ETV6 as a pro-viral factor in host response by inhibiting TBK1 phosphorylation, furthering our understandings of RNA virus immunosuppression and providing a valuable clue to the development of antiviral reagents for the control of avian RNA virus infection.

Gene silencing in broomrapes and other parasitic plants of the Orobanchaceae family: mechanisms, considerations, and future directions.

Holoparasites of the Orobanchaceae family are devastating pests causing severe damage to many crop species and are nearly impossible to control with conventional methods. During past decades RNA interference (RNAi) has been seen as a promising approach to control various crop pests. The exchange of small RNAs (sRNAs) between crops and parasitic plants has been documented indicating a potential for the development of methods to protect them via the delivery of the sRNAs to parasites, called host-induced gene silencing (HIGS). Here we describe various approaches used for gene silencing in plants and suggest solutions to improve the long-distance movement of the silencing triggers to elevate the HIGS efficiency in parasitic plants. We also investigate the important biological processes during parasites life cycle with a focus on broomrape species, providing several appropriate target genes that can be used in, especially, multiplex gene silencing experiments. We also touch on how the application of nanoparticles can improve the stability and delivery of the silencing triggers, highlighting its potential for parasitic plants control. Finally, suggestions for further research and possible directions for RNAi in parasitic plants are provided.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.

Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a disease-causing mechanism.

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3'-untranslated region (3'-UTR) of SPINK1 as the cause of severe infantile isolated exocrine pancreatic insufficiency. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here, we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that, owing to its particular location, Alu_Ins could independently disrupt mRNA 3' end formation and/or affect other post-transcriptional processes such as nuclear export and translation. However, employing a 3'-UTR luciferase reporter assay, Alu_Ins was found to result in only an ∼50% reduction in luciferase activity compared to wild type, which is insufficient to account for the severe pancreatic deficiency in the Alu_Ins homozygote. We then postulated that double-stranded RNA (dsRNA) structures formed between Alu elements, an upstream mechanism regulating gene expression, might be responsible. Using RepeatMasker, we identified two Alu elements within SPINK1's third intron, both oriented oppositely to Alu_Ins. Through RNAfold predictions and full-length gene expression assays, we investigated orientation-dependent interactions between these Alu repeats. We provide compelling evidence to link the detrimental effect of Alu_Ins to extensive dsRNA structures formed between Alu_Ins and pre-existing intronic Alu sequences, including the restoration of SPINK1 mRNA expression by aligning all three Alu elements in the same orientation. Given the widespread presence of Alu elements in the human genome and the potential for new Alu insertions at almost any locus, our findings have important implications for detecting and interpreting Alu insertions in disease genes.

The 5-Fluorouracil RNA Expression Viewer (5-FUR) Facilitates Interpreting the Effects of Drug Treatment and RRP6 Deletion on the Transcriptional Landscape in Yeast.

Saccharomyces cerevisiae is an excellent model to study the effect of external cues on cell division and stress response. 5-Fluorocuracil (5-FU) has been used to treat solid tumors since several decades. The drug was initially designed to interfere with DNA replication but was later found to exert its antiproliferative effect also via RNA-dependent processes. Since 5-FU inhibits the activity of the 3'-5'-exoribonuclease Rrp6 in yeast and mammals, earlier work has compared the effect of 5-FU treatment and RRP6 deletion at the transcriptome level in diploid synchronized yeast cells. To facilitate interpreting the expression data we have developed an improved 5-Fluorouracil RNA (5-FUR) expression viewer. Users can access information via genome coordinates and systematic or standard names for mRNAs and Xrn1-dependent-, stable-, cryptic-, and meiotic unannotated transcripts (XUTs, SUTs, CUTs, and MUTs). Normalized log2-transformed or linear data can be displayed as filled diagrams, line graphs or color-coded heatmaps. The expression data are useful for researchers interested in processes such as cell cycle regulation, mitotic repression of meiotic genes, the effect of 5-FU treatment and Rrp6 deficiency on the transcriptome and expression profiles of sense/antisense loci that encode overlapping transcripts. The viewer is accessible at http://5fur.genouest.org.

Silence is not always golden: A closer look at potential environmental and ecotoxicological impacts of large-scale dsRNA application.

RNA interference (RNAi) technology has emerged as a pivotal strategy in sustainable pest management, offering a targeted approach that significantly mitigates the environmental and health risks associated with traditional insecticides. Originally implemented through genetically modified organisms (GMOs) to produce specific RNAi constructs, the technology has evolved in response to public and regulatory concerns over GMOs. This evolution has spurred the development of non-transgenic RNAi applications such as spray-induced gene silencing (SIGS), which employs double-stranded RNA (dsRNA) to silence pest genes directly without altering the plant's genetic makeup. Despite its advantages in specificity and reduced ecological footprint, SIGS faces significant obstacles, particularly the instability of dsRNA in field conditions, which limits its practical efficacy. To overcome these limitations, innovative delivery mechanisms have been developed. These include nanotechnology-based systems, minicells, and nanovesicles, which are designed to protect dsRNA from degradation and enhance its delivery to target organisms. While these advancements have improved the stability and application efficiency of dsRNA, comprehensive assessments of their environmental safety and the potential for increased exposure risks to non-target organisms remain incomplete. This comprehensive review aims to elucidate the environmental fate of dsRNA and evaluate the potential risks associated with its widespread application on non-target organisms, encompassing soil microorganisms, beneficial insects, host plants, and mammals. The objective is to establish a more refined framework for RNAi risk assessment within environmental and ecotoxicological contexts, thereby fostering the development of safer, non-transgenic RNAi-based pest control strategies.

ISGylation enhances dsRNA-induced interferon response and NFκB signaling in fallopian tube epithelial cells.

Heritable mutations in BRCA1 associate with increased risk of high-grade serous tubo-ovarian cancer. Nongenetic risk factors associated with this cancer, which arises from fallopian tube epithelial (FTE) cells, suggests a role for repetitive ovulation wherein FTE cells are exposed to inflammatory signaling molecules within follicular fluid. We previously reported increased NFκB and EGFR signaling in BRCA1-deficient primary FTE cells, with follicular fluid exposure further increasing abundance of interferon-stimulated gene (ISG) transcripts, including the ubiquitin-like protein ISG15 and other ISGylation pathway members. Both NFκB and type I interferon signaling are upregulated by stimulation of cGAS-STING or MDA5 and RIGI pattern recognition receptors. Since some pattern recognition receptors and their signal transduction pathway members are ISGylated, we tested the impact of ISG15 and ISGylation on interferon regulatory factor 3 (IRF3) and NFκB signaling through cGAS-STING or RIGI and MDA5 activation. Expression of ISG15 or UBA7, the E1-like ISG15-activating enzyme, in immortalized FTE cells was disrupted by CRISPR gene editing. Activation of IRF3 by RIGI or MDA5 but not cGAS-STING was attenuated by loss of either ISG15 or UBA7 and this was reflected by a similar effect on NFκB activation and downstream targets. Loss of ISGylation decreased levels of both MDA5 and RIGI, with knockdown of RIGI but not MDA5, decreasing IRF3 and NFκB activation in parental cells. These finding indicate that ISGylation enhances the ability of dsRNA to activate cytokine release and proinflammatory signaling. Further work to explore ISGylation as a target for prevention of high-grade serous tubo-ovarian cancer in BRCA1 mutation carriers is warranted.

The genome sequencing and comparative genomics analysis of Rhizoctonia solani reveals a novel effector family owning a uinque domain in Basidiomycetes.

Rhizoctonia solani is a soil-borne pathogen with 14 anastomosis groups (AGs), and different subgroups are genetically diverse. However, the genetic factors contributing to the pathogenicity of the fungus have not been well characterized. In this study, the genome of R. solani AG1-ZJ was sequenced. As the result, a 41.57 Mb draft genome containing 12,197 putative coding genes was obtained. Comparative genomic analysis of 11 different AGs revealed conservation and unique characteristics between the AGs. Furthermore, a novel effector family containing a 68 amino acid conserved domain unique in basidiomycetous fungi was characterized. Two effectors containing the conserved domain in AG4-JY were identified, and named as RsUEB1 and RsUEB2. Furthermore, the spray-induced gene silencing strategy was used to generate a dsRNA capable of silencing the conserved domain sequence of RsUEB1 and RsUEB2. This dsRNA can significantly reduce the expression of RsUEB1 and RsUEB2 and the pathogenicity of AG4-JY on foxtail millet, maize, rice and wheat. In conclusion, this study provides significant insights into the pathogenicity mechanisms of R. solani. The identification of the conserved domain and the successful use of dsRNA silencing of the gene containing the conserved domain will offer a new strategy for controlling sheath blight in cereal crops.

Ex vivo delivery of dsRNA targeting ryanodine receptors for control of Tuta absoluta.

BACKGROUND: RNA interference (RNAi) is an endogenous eukaryote viral defence mechanism representing a unique form of post-transcriptional gene silencing. Owing to its high specificity, this technology is being developed for use in dsRNA-based biopesticides for control of pest insects. Whilst many lepidopteran species are recalcitrant to RNAi, Tuta absoluta, a polyphagous insect responsible for extensive crop damage, is sensitive. Ryanodine receptors (RyRs) are intracellular calcium channels regulating calcium ion (Ca2+) release. The chemical pesticide class of diamides functions agonistically against lepidopteran RyR, resulting in uncontrolled Ca2+ release, feeding cessation and death. Resistance to diamides has emerged in T. absoluta, derived from RyR point mutations. RESULTS: RNAi was used to target RyR transcripts of T. absoluta. Data presented here demonstrate the systemic use of exogenous T. absoluta RyR-specific (TaRy) dsRNA in tomato plants (Solanum lycopersicum) to significantly downregulate expression of the target gene, resulting in significant insect mortality and reduced leaf damage. Using a leaflet delivery system, daily dosing of 3 µg TaRy dsRNA for 72 h resulted in 50% downregulation of the target gene and 50% reduction in tomato leaf damage. Corrected larval mortality and adult emergence were reduced by 38% and 33%, respectively. TaRy dsRNA demonstrated stability in tomato leaves ≤72 h after dosing. CONCLUSIONS: This work identifies TaRy as a promising target for RNAi control of this widespread crop pest. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

RNA interference targeting WSSV ribonucleotide reductase 2 provides long-term protection against infection in Litopenaeus vannamei.

Many studies have demonstrated that long double-stranded RNAs (dsRNAs) targeting essential genes of white spot syndrome virus (WSSV) can induce a sequence-specific antiviral RNA interference (RNAi) response in shrimp, thereby offering protection against WSSV infection. However, further experimental data on the required dose of dsRNAs and the duration of protection from a single administration are necessary to establish RNAi-mediated methods as effective and practical antiviral measures. In this study, we evaluated the protective efficacy and the duration of protection provided by a single administration of various doses of long dsRNA targeting WSSV ribonucleotide reductase 2 (rr2) in white-leg shrimp Litopenaeus vannamei. The protective efficacy of long dsRNA targeting WSSV rr2 was not diminished by the reduction of the dose to 100 ng g-1 of body weight, suggesting that a relatively low dose can effectively induce an RNAi response in shrimp. Furthermore, shrimp were well-protected against WSSV challenges for up to 4 wk post-administration of the rr2-targeting long dsRNA, although the protective effect almost disappeared at 6 wk post-administration. These results suggest that long dsRNAs can provide protection against WSSV for at least 1 mo, and monthly administration of long dsRNAs could serve as a long-term protective strategy for shrimp against WSSV.

Identification of whole-cell dsRNA-binding proteins by phase separation.

Interactions between double-stranded RNA (dsRNA) and proteins play an important role in cellular homeostasis by regulating the editing, stability, and splicing of intracellular RNA. The identification of dsRNA-binding proteins (dsRBPs) is key; however, it has long been challenging to purify dsRBPs from cells. In this study, we developed a novel method, dsRBPC (dsRNA-binding protein capture), to purify cellular dsRBPs based on classic phase separation purification procedures. A global dsRNA-binding proteome of LLC-PK1 cells was obtained, and we identified 1326 dsRBPs, including 1303 putative novel dsRBPs. Functional analyses suggested that these enriched dsRBPs are mainly associated with rRNA processing, RNA splicing, transcriptional regulation, and nucleocytoplasmic transport. We also found that the ARM (armadillo/beta-catenin-like repeats) motif is a previously unknown dsRNA-binding domain, as demonstrated by biochemical experiments. Collectively, this study provides a useful approach for dsRBP identification and the discovery of a global dsRNA-binding proteome to comprehensively map the dsRNA - protein interaction network.

Development and Application of Automated Sandwich ELISA for Quantitating Residual dsRNA in mRNA Vaccines.

The rise of mRNA as a novel vaccination strategy presents new opportunities to confront global disease. Double-stranded RNA (dsRNA) is an impurity byproduct of the in vitro transcription reaction used to manufacture mRNA that may affect the potency and safety of the mRNA vaccine in patients. Careful quantitation of dsRNA during manufacturing is critical to ensure that residual dsRNA is minimized in purified mRNA drug substances. In this work, we describe the development and implementation of a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) to quantitate nanogram quantities of residual dsRNA contaminants in mRNA process intermediates using readily available commercial reagents. This sandwich ELISA developed in this study follows a standard protocol and can be easily adapted to most research laboratory environments. Additionally, a liquid handler coupled with an automated robotics system was utilized to increase assay throughput, improve precision, and reduce the analyst time requirement. The final automated sandwich ELISA was able to measure <10 ng/mL of dsRNA with a specificity for dsRNA over 2000-fold higher than mRNA, a variability of <15%, and a throughput of 72 samples per day.