Modeling recursive RNA interference.

An important application of the RNA interference (RNAi) pathway is its use as a small RNA-based regulatory system commonly exploited to suppress expression of target genes to test their function in vivo. In several published experiments, RNAi has been used to inactivate components of the RNAi pathway itself, a procedure termed recursive RNAi in this report. The theoretical basis of recursive RNAi is unclear since the procedure could potentially be self-defeating, and in practice the effectiveness of recursive RNAi in published experiments is highly variable. A mathematical model for recursive RNAi was developed and used to investigate the range of conditions under which the procedure should be effective. The model predicts that the effectiveness of recursive RNAi is strongly dependent on the efficacy of RNAi at knocking down target gene expression. This efficacy is known to vary highly between different cell types, and comparison of the model predictions to published experimental data suggests that variation in RNAi efficacy may be the main cause of discrepancies between published recursive RNAi experiments in different organisms. The model suggests potential ways to optimize the effectiveness of recursive RNAi both for screening of RNAi components as well as for improved temporal control of gene expression in switch off-switch on experiments

Effects of Nanoparticles on Double-Stranded RNA Stability in Moth Hemolymph

RNA interference (RNAi) is an immune response in which double-stranded RNA (dsRNA) suppresses a target gene. By designing dsRNA to target genes that are necessary for life, dsRNA can potentially be used as an insecticide. RNAi-based insecticides are badly needed because they are more specific than conventional pesticides and because many insects have developed resistance to pesticides. Unfortunately, some insects produce enzymes that degrade dsRNA and prevent the RNAi response (Cooperet al., 2018). Therefore, RNAi-based insecticides currently cannot be used to control all insects. Here we investigate dsRNA stability when incubated in hemolymph ex vivoto determine if degradation of dsRNA is contributing to the inadequate RNAi response exhibited by lepidopterans, such as the European corn borer (ECB, Ostrinia nubilalis). Our findings indicate that dsRNA is significantly degraded in ECB hemolymph, but encapsulation of dsRNA in chitosan-based nanoparticles (CB-NPs) enhances stability. These findings provide insight into RNAi efficiency limitations in insects, and may provide a method to enhance RNAi efficiency in lepidopterans and other RNAi-refractory pests

Suppression of Argonaute 2 Transcript Levels in Du182A Cells

RNA interference (RNAi) uses double-stranded RNA (dsRNA) molecules to degrade and suppress the transcript level of a complementary mRNA target1. The RNAi pathway is complex and includes many different proteins, like argonautes, in the core machinery. Argonautes are dsRNA binding proteins which help recognize and cleave target mRNA molecules. In our experiments, we attempted to suppress the transcript level of argonaute 2 (Ago2) in a Diabrotica undecimpunctata cell line (Du182A) using dsRNA, with the idea of disrupting the RNAi pathway using an RNAi of RNAi technique. Ago2 transcript levels were suppressed following treatment with dsRNA. Future experiments can now use this technique, with some modification to better understand the RNAi pathway

RNAi efficiency, systemic properties, and novel delivery methods for pest insect control : what we know so far

In recent years, the research on the potential of using RNA interference (RNAi) to suppress crop pests has made an outstanding growth. However, given the variability of RNAi efficiency that is observed in many insects, the development of novel approaches toward insect pest management using RNAi requires first to unravel factors behind the efficiency of dsRNA-mediated gene silencing. In this review, we explore essential implications and possibilities to increase RNAi efficiency by delivery of dsRNA through non-transformative methods. We discuss factors influencing the RNAi mechanism in insects and systemic properties of dsRNA. Finally, novel strategies to deliver dsRNA are discussed, including delivery by symbionts, plant viruses, trunk injections, root soaking, and transplastomic plants

Israeli acute paralysis virus infection leads to an enhanced RNA interference response and not its suppression in the bumblebee Bombus terrestris

RNA interference (RNAi) is the primary antiviral defense system in insects and its importance for pollinator health is indisputable. In this work, we examined the effect of Israeli acute paralysis virus (IAPV) infection on the RNAi process in the bumblebee, Bombus terrestris, and whether the presence of possible functional viral suppressors could alter the potency of the host's immune response. For this, a two-fold approach was used. Through a functional RNAi assay, we observed an enhancement of the RNAi system after IAPV infection instead of its suppression, despite only minimal upregulation of the genes involved in RNAi. Besides, the presence of the proposed suppressor 1A and the predicted OrfX protein in IAPV could not be confirmed using high definition mass spectrometry. In parallel, when bumblebees were infected with cricket paralysis virus (CrPV), known to encode a suppressor of RNAi, no increase in RNAi efficiency was seen. For both viruses, pre-infection with the one virus lead to a decreased replication of the other virus, indicating a major effect of competition. These results are compelling in the context of Dicistroviridae in multi-virus/multi-host networks as the effect of a viral infection on the RNAi machinery may influence subsequent virus infections

Postembryonic RNAi in Heterorhabditis bacteriophora: a nematode insect parasite and host for insect pathogenic symbionts

Background: Heterorhabditis bacteriophora is applied throughout the world for the biological control of insects and is an animal model to study interspecies interactions, e.g. mutualism, parasitism and vector-borne disease. H. bacteriophora nematodes are mutually associated with the insect pathogen, Photorhabdus luminescens. The developmentally arrested infective juvenile (IJ) stage nematode (vector) specifically transmits Photorhabdus luminescens bacteria (pathogen) in its gut mucosa to the haemocoel of insects (host). The nematode vector and pathogen alone are not known to cause insect disease. RNA interference is an excellent reverse genetic tool to study gene function in C. elegans, and it would be useful in H. bacteriophora to exploit the H. bacteriophora genome project, currently in progress. Results: Soaking L1 stage H. bacteriophora with seven dsRNAs of genes whose C. elegans orthologs had severe RNAi phenotypes resulted in highly penetrant and obvious developmental and reproductive abnormalities. The efficacy of postembryonic double strand RNA interference (RNAi) was evident by abnormal gonad morphology and sterility of adult H. bacteriophora and C. elegans presumable due to defects in germ cell proliferation and gonad development. The penetrance of RNAi phenotypes in H. bacteriophora was high for five genes (87-100%; Hba-cct-2, Hba-daf-21, Hba-icd-1; Hba-nol-5, and Hba-W01G7.3) and moderate for two genes (usually 30-50%; Hba-rack-1 and Hba-arf-1). RNAi of three additional C. elegans orthologs for which RNAi phenotypes were not previously detected in C. elegans, also did not result in any apparent phenotypes in H. bacteriophora. Specific and severe reduction in transcript levels in RNAi treated L1s was determined by quantitative real-time RT-PCR. These results suggest that postembryonic RNAi by soaking is potent and specific. Conclusion: Although RNAi is conserved in animals and plants, RNAi using long dsRNA is not. These results demonstrate that RNAi can be used effectively in H. bacteriophora and can be applied for analyses of nematode genes involved in symbiosis and parasitism. It is likely that RNAi will be an important tool for functional genomics utilizing the high quality draft H. bacteriophora genome sequence

RNA interference approaches for treatment of HIV-1 infection.

HIV/AIDS is a chronic and debilitating disease that cannot be cured with current antiretroviral drugs. While combinatorial antiretroviral therapy (cART) can potently suppress HIV-1 replication and delay the onset of AIDS, viral mutagenesis often leads to viral escape from multiple drugs. In addition to the pharmacological agents that comprise cART drug cocktails, new biological therapeutics are reaching the clinic. These include gene-based therapies that utilize RNA interference (RNAi) to silence the expression of viral or host mRNA targets that are required for HIV-1 infection and/or replication. RNAi allows sequence-specific design to compensate for viral mutants and natural variants, thereby drastically expanding the number of therapeutic targets beyond the capabilities of cART. Recent advances in clinical and preclinical studies have demonstrated the promise of RNAi therapeutics, reinforcing the concept that RNAi-based agents might offer a safe, effective, and more durable approach for the treatment of HIV/AIDS. Nevertheless, there are challenges that must be overcome in order for RNAi therapeutics to reach their clinical potential. These include the refinement of strategies for delivery and to reduce the risk of mutational escape. In this review, we provide an overview of RNAi-based therapies for HIV-1, examine a variety of combinatorial RNAi strategies, and discuss approaches for ex vivo delivery and in vivo delivery

The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed

RNA interference knockdown of BRASSINOSTEROID INSENSITIVE1 in maize reveals novel functions for brassinosteroid signaling in controlling plant architecture

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbril complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbriI-RNAi transgenic lines have compromised BR signaling. zmbriI-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbriI-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbriI-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbriI-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling