Trigger RNA Interference Research Articles

Delivery of short hairpin RNA in the neotropical brown stink bug, Euschistus heros, using a composite nanomaterial

The response of insects to orally delivered double-stranded RNA ranges widely among taxa studied to date. Long dsRNA does elicit a response in stink bugs but the dose required to achieve an effect is relatively high compared to other insects such Colorado potato beetle or western corn rootworm. Improving the delivery of dsRNA to stink bugs will improve the likelihood of using RNA-based biocontrols for the management of these economically important pests. Short hairpin RNA (shRNA) is a useful molecule with which to test improvements in the delivery of double stranded RNA in the neotropical brown stink bug, Euschistus heros, since shRNA alone does not elicit a clear effect like that for long dsRNA. Here, we show for the first time the oral delivery of shRNA triggering RNA interference (RNAi) in E. heros using 4 nm cerium oxide nanoparticles (CeO2 NPs) coated with diethylamioethyl dextran (Dextran-DEAE) as a carrier. We identified particle properties (coating composition and degree of substitution, hydrodynamic diameter, and zeta potential) and shRNA loading rates (Ce:shRNA mass ratio) that resulted in successful transcript reduction or RNAi. When the Z-average diameter of CeO2 Dextran-DEAE-shRNA NP complex was less than 250 nm and the zeta potential was in the 15–25 mV range (Ce:shRNA mass ratio of 0.7:1), significant mortality attributed to RNAi was observed with a shRNA concentration in feeding solution of 250 ng/μl. The degradation of the targeted troponin transcript by NP-delivered shRNA was equivalent to that observed with long dsRNA, while naked shRNA transcript reduction was not statistically significant. Elemental mapping by synchrotron X-ray fluorescence microprobe confirmed uptake and distribution of Ce throughout the body with the highest concentrations found in gut tissue. Taken together, our results suggest that a nanoparticle delivery system can improve the delivery of RNA-based biocontrols to E. heros, and therefore its attractiveness as an application in the management of this important pest in soybean production.

Induction of Promoter DNA Methylation Upon High-Pressure Spraying of Double-Stranded RNA in Plants

Exogenous application of RNA molecules is a potent method to trigger RNA interference (RNAi) in plants in a transgene-free manner. So far, all exogenous RNAi (exo-RNAi) applications have aimed to trigger mRNA degradation of a given target. However, the issue of concomitant epigenetic changes was never addressed. Here, we report for the first time that high-pressure spraying of dsRNAs can trigger de novo methylation of promoter sequences in plants.

Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in Arabidopsis thaliana.

Recent studies have revealed that foliar application of double-stranded RNAs (dsRNAs) or small-interfering RNAs (siRNAs) encoding specific genes of plant pathogens triggered RNA interference (RNAi)-mediated silencing of the gene targets. However, a limited number of reports documented silencing of plant endogenes or transgenes after direct foliar RNA application. This study analyzed the importance of physiological conditions (plant age, time of day, soil moisture, high salinity, heat, and cold stresses) and different dsRNA application means (brush spreading, spraying, infiltration, inoculation, needle injection, and pipetting) for suppression of neomycin phosphotransferase II (NPTII) transgene in Arabidopsis thaliana, as transgenes are more prone to silencing. We observed a higher NPTII suppression when dsRNA was applied at late day period, being most efficient at night, which revealed a diurnal variation in dsRNA treatment efficacy. Exogenous NPTII-dsRNA considerably reduced NPTII expression in 4-week-old plants and only limited it in 2- and 6-week-old plants. In addition, a more discernible NPTII downregulation was detected under low soil moisture conditions. Treatment of adaxial and abaxial leaf surfaces by brushes, spraying, and pipetting showed a higher NPTII suppression, while infiltration and inoculation were less efficient. Thus, appropriate plant age, late time of day, low soil moisture, and optimal dsRNA application modes are important for exogenously induced gene silencing.

Artificial miRNAs as therapeutic tools: Challenges and opportunities.

RNA interference (RNAi) technology has been used for almost two decades to study gene functions and in therapeutic approaches. It uses cellular machinery and small, designed RNAs in the form of synthetic small interfering RNAs (siRNAs) or vector-based short hairpin RNAs (shRNAs), and artificial miRNAs (amiRNAs) to inhibit a gene of interest. Artificial miRNAs, known also as miRNA mimics, shRNA-miRs, or pri-miRNA-like shRNAs have the most complex structures and undergo two-step processing in cells to form mature siRNAs, which are RNAi effectors. AmiRNAs are composed of a target-specific siRNA insert and scaffold based on a natural primary miRNA (pri-miRNA). siRNAs serve as a guide to search for complementary sequences in transcripts, whereas pri-miRNA scaffolds ensure proper processing and transport. The dynamics of siRNA maturation and siRNA levels in the cell resemble those of endogenous miRNAs; therefore amiRNAs are safer than other RNAi triggers. Delivered as viral vectors and expressed under tissue-specific polymerase II (Pol II) promoters, amiRNAs provide long-lasting silencing and expression in selected tissues. Therefore, amiRNAs are useful therapeutic tools for a broad spectrum of human diseases, including neurodegenerative diseases, cancers and viral infections. Recent reports on the role of sequence and structure in pri-miRNA processing may contribute to the improvement of the amiRNA tools. In addition, the success of a recently initiated clinical trial for Huntington's disease could pave the way for other amiRNA-based therapies, if proven effective and safe. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action RNA in Disease and Development > RNA in Disease.

Leading RNA Interference Therapeutics Part 2: Silencing Delta-Aminolevulinic Acid Synthase 1, with a Focus on Givosiran.

In November 2019 givosiran became the second small interfering RNA (siRNA)-based drug to receive US Food and Drug Administration (FDA) approval, it has been developed for the treatment of acute intermittent porphyria (AIP), a disorder characterized by life-threatening acute neurovisceral attacks. Theporphyrias are a group of disorders in which enzymatic deficiencies in heme production lead to toxic accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG), which are involved in the neurovisceral attacks. Givosiran acts as a conventional siRNA to trigger RNA interference (RNAi)-mediated gene silencing on delta-ALA synthase 1 (ALAS1), thus returning ALA and PBG metabolites to the physiological level to attenuate further neurotoxicity. Givosiran makes use of a new hepatic-delivery system that conjugates three GalNac (N-acetylgalactosamine) molecules to the siRNA passenger strand. GalNac binds to the liver asialoglycoprotein receptor, favoring the internalization of these GalNac-conjugated siRNAs into the hepatic cells. In a phase I study, subcutaneous monthly administration of givosiran 2.5mg/kg reduced > 90% of ALA and PBG content. This siRNA is being analyzed in ENVISION (NCT03338816), a phase III, multicenter, placebo-controlled randomized controlled trial. In preliminary results, givosiran achieved clinical endpoints for AIP, reducing urinary ALA levels, and presented a safety profile that enabled further drug development. The clinical performance of givosiran revealed that suppression of ALAS1 by GalNac-decorated siRNAs represents an additional approach for the treatment of patients with AIP that manifests recurrent acute neurovisceral attacks.

Tripodal RNA with 3' Overhangs Prevents RIG-I-Mediated Innate Immune Response.

RNA interference (RNAi) offers great promise in life science research and therapeutic development, as it easily achieves a potent target gene knockdown with high specificity. Since the conventional small interfering RNA (siRNA) structure, known as 19 bp double-stranded RNA (dsRNA) with 2-nucleotide (nt) 3' overhang, has been introduced to successfully elicit the RNAi in mammalian cells, a variety of structural variants of RNAi trigger have been developed. Our group previously reported branched, tripodal interfering RNA (tiRNA) structures as a multigene targeting RNA structure inducing RNAi. However, the immune stimulatory effect of branched tiRNA structure has not been thoroughly evaluated. In this study, we show that tiRNA with blunt ends triggers innate immune response in T98G cell and mouse macrophage cells, which is dependent upon the retinoic acid-inducible gene I (RIG-I), a well-known cytoplasmic dsRNA sensor. Interestingly, immune response triggered by tiRNA can be suppressed by the introduction of 2-nt 3' overhang structure. Our finding expands the structural diversity of RIG-I ligands and provides a guide to develop a safe multitargeting RNA structure for therapeutic application.

Gene Silencing: The Mechanism to Down Regulate the Target Gene

Gene silencing is one of the most efficient and promising functional genomics tools which down regulates the expression of a gene in a very precise manner and has significant impact on crop improvement. Silencing of a target gene can be achieved at two levels; transcriptional and post-transcriptional stages. In broader sense, transcriptional gene silencing occurs through the repression of the process of transcription while the post transcriptional gene silencing can occur through the degradation of the mRNA. Small RNA molecules like siRNAs & miRNAs are key players which mediate the mechanism of gene silencing. These small RNA molecules are generated through Dicer digestion of the endogenous or exogenous dsRNA by which RISC (RNA induced gene silencing complex) is activated which inhibits expression of a gene. Apart from these small RNA molecules, viruses can also accomplish gene silencing which is known as VIGS (Virus induced gene silencing). VIGS consists of cloning and inserting plant endogenous gene sequences in recombinant viral vectors, which are then inoculated in plants, triggering RNA interference. Gene silencing as reverse genetic tool, has been exploited successfully to build up the resistant against biotic (insect/pest, nematodes, viruses etc.) and abiotic stresses (drought, salinity, frost etc.), and for the enhancement of yield and quality parameters. With the development of cutting edge biotechnological tools and techniques, it is now affordable to knock down/down regulate the expression of any undesirable gene. Ultimately this kind of technology can be important to global food security and sustainability.

Dicer-2 Regulates Resistance and Maintains Homeostasis against Zika Virus Infection in Drosophila.

Zika virus (ZIKV) outbreaks pose a massive public health threat in several countries. We have developed an in vivo model to investigate the host-ZIKV interaction in Drosophila We have found that a strain of ZIKV replicates in wild-type flies without reducing their survival ability. We have shown that ZIKV infection triggers RNA interference and that mutating Dicer-2 results in enhanced ZIKV load and increased susceptibility to ZIKV infection. Using a flavivirus-specific Ab, we have found that ZIKV is localized in the gut and fat body cells of the infected wild-type flies and results in their perturbed homeostasis. In addition, Dicer-2 mutants display severely reduced insulin activity, which could contribute toward the increased mortality of these flies. Our work establishes the suitability of Drosophila as the model system to study host-ZIKV dynamics, which is expected to greatly advance our understanding of the molecular and physiological processes that determine the outcome of this disease.

Delivery of miRNA-Targeted Oligonucleotides in the Rat Striatum by Magnetofection with Neuromag®.

MicroRNAs (miRNAs) regulate gene expression at posttranscriptional level by triggering RNA interference. In such a sense, aberrant expressions of miRNAs play critical roles in the pathogenesis of many disorders, including Parkinson’s disease (PD). Controlling the level of specific miRNAs in the brain is thus a promising therapeutic strategy for neuroprotection. A fundamental need for miRNA regulation (either replacing or inhibition) is a carrier capable of delivering oligonucleotides into brain cells. This study aimed to examine a polymeric magnetic particle, Neuromag®, for delivery of synthetic miRNA inhibitors in the rat central nervous system. We injected the miRNA inhibitor complexed with Neuromag® into the lateral ventricles next to the striatum, by stereotaxic surgery. Neuromag efficiently delivered oligonucleotides in the striatum and septum areas, as shown by microscopy imaging of fluorescein isothiocyanate (FITC)-labeled oligos in astrocytes and neurons. Transfected oligos showed efficacy concerning miRNA inhibition. Neuromag®-structured miR-134 antimiR (0.36 nmol) caused a significant 0.35 fold decrease of striatal miR-134, as revealed by real-time quantitative polymerase chain reaction (RT-qPCR). In conclusion, the polymeric magnetic particle Neuromag® efficiently delivered functional miRNA inhibitors in brain regions surrounding lateral ventricles, particularly the striatum. This delivery system holds potential as a promising miRNA-based disease-modifying drug and merits further pre-clinical studies using animal models of PD.

Synthetic biology approach for plant protection using dsRNA.

SummaryPathogens induce severe damages on cultivated plants and represent a serious threat to global food security. Emerging strategies for crop protection involve the external treatment of plants with double‐stranded (ds)RNA to trigger RNA interference. However, applying this technology in greenhouses and fields depends on dsRNA quality, stability and efficient large‐scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo dsRNA production system in Pseudomonas syringae bacteria. Unlike other in vitro or in vivo dsRNA production systems that rely on DNA transcription and postsynthetic alignment of single‐stranded RNA molecules, the phi6 system is based on the replication of dsRNA by an RNA‐dependent RNA polymerase, thus allowing production of high‐quality, long dsRNA molecules. The phi6 replication complex was reprogrammed to multiply dsRNA sequences homologous to tobacco mosaic virus (TMV) by replacing the coding regions within two of the three phi6 genome segments with TMV sequences and introduction of these constructs into P. syringae together with the third phi6 segment, which encodes the components of the phi6 replication complex. The stable production of TMV dsRNA was achieved by combining all the three phi6 genome segments and by maintaining the natural dsRNA sizes and sequence elements required for efficient replication and packaging of the segments. The produced TMV‐derived dsRNAs inhibited TMV propagation when applied to infected Nicotiana benthamiana plants. The established dsRNA production system enables the broad application of dsRNA molecules as an efficient, highly flexible, nontransgenic and environmentally friendly approach for protecting crops against viruses and other pathogens.

Natural Antisense Transcripts at the Interface between Host Genome and Mobile Genetic Elements.

Non-coding RNAs are involved in epigenetic processes, playing a role in the regulation of gene expression at the transcriptional and post-transcriptional levels. A particular group of ncRNA are natural antisense transcripts (NATs); these are transcribed in the opposite direction to protein coding transcripts and are widespread in eukaryotes. Their abundance, evidence of phylogenetic conservation and an increasing number of well-characterized examples of antisense-mediated gene regulation are indicative of essential biological roles of NATs. There is evidence to suggest that they interfere with their corresponding sense transcript to elicit concordant and discordant regulation. The main mechanisms involved include transcriptional interference as well as dsRNA formation. Sense–antisense hybrid formation can trigger RNA interference, RNA editing or protein kinase R. However, the exact molecular mechanisms elicited by NATs in the context of these regulatory roles are currently poorly understood. Several examples confirm that ectopic expression of antisense transcripts trigger epigenetic silencing of the related sense transcript. Genomic approaches suggest that the antisense transcriptome carries a broader biological significance which goes beyond the physiological regulation of the directly related sense transcripts. Because NATs show evidence of conservation we speculate that they played a role in evolution, with early eukaryotes gaining selective advantage through the regulatory effects. With the surge of genome and transcriptome sequencing projects, there is promise of a more comprehensive understanding of the biological role of NATs and the regulatory mechanisms involved.

Evaluation of potential RNA-interference-target genes to control cotton mealybug, Phenacoccus solenopsis (Hemiptera: Pseudococcuidae).

RNA interference (RNAi) of vital insect genes is a potential tool for targeted pest control. However, selection of the right target genes is a challenge because the RNAi efficacy is known to vary among insect species. Cotton mealybug, Phenacoccus solenopsis, is a phloem-feeding economically important crop pest. We evaluated the RNAi of 2 vital genes, Bursicon (PsBur) and V-ATPase (PsV-ATPase) as potential targets in P. solenopsis for its control. PCR fragments of PsBur and PsV-ATPase were amplified using cDNA synthesized from the total RNA. The PCR amplicons were cloned into Potato virus X (PVX) to develop recombinant PVX for the inoculation of Nicotiana tabacum plants for bioassays with healthy P. solenopsis. Reverse-transcription-polymerase chain reaction (RT-PCR) was used to validate the expression of transgenes in the recombinant-PVX-inoculated plants (treated), and suppression of the target genes in the mealybugs exposed to them. The RT-PCR confirmed the expression of transgenes in the treated plants. Mealybug individuals on treated plants either died or showed physical deformities. Further, the population of mealybug was significantly reduced by feeding on N. tabacum expressing RNAi triggers against PsBur and PsV-ATPase. The results conclude that RNAi is activated in P. solenopsis by feeding on N. tabacum expressing RNAi triggering elements of PsBur and PsV-ATPase genes through recombinant PVX vector. Further, V-ATPase and Bursicon genes are potential targets for RNAi-mediated control of P. solenopsis.

Abstract 36: Factor XII RNAi-based Therapeutic as a Prophylactic Anti-thrombotic Therapy

Introduction: Current anticoagulants effectively prevent thromboembolic (TBE) events, but unmet medical need remains due to elevated risk of major bleeding events, particularly where Factor Xa inhibitors are contraindicated. Factor XII (F12) zymogen initiates the intrinsic coagulation pathway through both autocleavage and Factor XI cleavage. F12 deficient mice show protection from induced thrombosis without increased bleeding risk. F12 deficiency in humans is not associated with increased bleeding risk, suggesting F12 is not essential for hemostasis. Methods: Hepatocyte-targeted RNA interference (RNAi) triggers for reducing liver F12 expression were developed for subcutaneous (SQ) administration. Specific rodent/human/non-human primate (NHP) cross-reactive RNAi triggers were designed in silico and screened for knockdown activity in vitro and in wild-type mice. Structure activity relationship (SAR) studies of active RNAi triggers identified optimal modifications for lead identification. Candidate RNAi triggers were evaluated for activity and safety in NHPs. In addition, candidate RNAi triggers were evaluated for activity in two rodent models of thrombosis and bleeding risk. Results: F12 RNAi triggers exhibited significant and sustained knockdown of serum F12 levels in both mice and NHPs. SAR studies enabled identification of a candidate lead molecule that demonstrated >95% knockdown after a single 1 mg/kg dose in mice. Evaluation of two SQ doses of this RNAi trigger in NHPs demonstrated >93% knockdown of serum F12 levels at nadir with durable knockdown of >90% for over 5 weeks after the final dose. Concomitant increases in coagulation biomarker aPTT were observed, consistent with functional F12 depletion. Both arteriovenous shunt studies in rats and FeCl 3 -induced TBE studies in mice exhibited prevention of clot formation after F12 knockdown. Importantly, F12 knockdown did not increase bleeding times in explicit hemostasis studies in mice. Conclusions: Development of a SQ-administered F12-specific RNAi trigger offers potential for a novel, infrequently dosed prophylactic treatment for TBE without increased bleeding risk. Administration by medical personnel would eliminate adherence concerns inherent to current therapies.

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses.

The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships.

Abstract 428: Lipoprotein(a) Targeting With RNAi Delivery Platforms in Transgenic Mice and Cynomolgus Monkeys

Background: Meta-analyses of clinical studies have demonstrated that high levels of lipoprotein(a), known as Lp(a), correlate with residual risk of cardiovascular disease (CVD) while Mendelian genetic studies indicate that Lp(a) is an independent cause of CVD. Lp(a) is a complex lipoprotein particle similar in structure to LDL (consisting of cholesterol, triglycerides, phospholipids and apoB-100), but contains the genetically variable liver-expressed protein apo(a). Lp(a) may contribute to atherosclerosis by targeting oxidized phospholipids to vascular injury sites, promoting both inflammation and accumulation of lipids. Methods: RNA interference (RNAi) based approaches for reducing liver apo(a) expression were developed using both a targeted intravenous platform called Dynamic Polyconjugates (DPC) TM and a subcutaneous (SQ) format. The DPC platform comprises an RNAi trigger conjugated to cholesterol, and a hepatocyte-targeted, membrane active peptide to promote endosomal release of the RNAi trigger. The SQ platform is an RNAi trigger targeted to liver by N-acetyl galactosamine in a proprietary format. RNAi activity was assessed in two different humanized transgenic mice and cynomolgus monkeys. Results: In Lp(a) transgenic mice expressing human apo(a) and apoB-100 genes, treatment with a single DPC dose reduced serum Lp(a) by >98% at nadir and >90% for >5 weeks. A single DPC injection in cynomolgus monkeys resulted in >95% reduction in endogenous Lp(a) particles at 5 weeks post injection, without significant changes in toxicity biomarkers. Treatment with a SQ RNAi trigger reduced serum apo(a) in transgenic mice by >90% at nadir, while that in monkeys reached 64% reduction of Lp(a). Additional modifications to the RNAi trigger improved both depth and duration of knockdown in transgenic mice. Conclusion: Long-duration reductions in circulating apo(a) and Lp(a) have been shown with hepatocyte-directed delivery of apo(a)-specific RNAi triggers in mice and monkeys. This represents an intriguing mechanism to evaluate in humans for reducing residual risk of CVD in LDL-controlled individuals with elevated Lp(a).

The involvement of clathrin-mediated endocytosis and two Sid-1-like transmembrane proteins in double-stranded RNA uptake in the Colorado potato beetle midgut.

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake.

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

Sequence-non-specific effects generated by various types of RNA interference triggers

RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.

Inoculation of Nicotiana tabacum with recombinant potato virus X induces RNA interference in the solenopsis mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

The chitin synthase 1 (CHS1) gene in Phenacoccus solenopsis (PsCHS1) was evaluated as a potential target of RNA interference (RNAi) by using Potato virus X (PVX) as a vector (recombinant PVX) for expressing RNAi triggering elements in Nicotiana tabacum L. RT-PCR analysis confirmed the expression of PsCHS1 in N. tabacum inoculated with recombinant-PVX-PsCHS1 (treated). RT- and multiplex-PCR further showed a reduction in mRNA levels of the target gene in mealybugs feeding on treated plants. Mortality in parent adults and emerging nymphs (21 and 29%) exposed to the treated plants was significantly higher (P < 0.05) than those exposed to uninoculated (-ve control) or inoculated with non-recombinant PVX (PVX-control). The number of surviving adults and the combined number of adults and nymphs (47 and 60%) was significantly (P < 0.05) lower on the treated plants than the -ve (76%) or PVX (74%) control. The visual observations verified the physical deformities in mealybugs exposed to the treated plants. chitin synthase 1 is a potential RNAi target in P. solenopsis and the recombinant PVX can be used as a tool to evaluate candidate RNAi triggering elements in plants.