siRNA Pathway Research Articles

MicroRNAs and other small RNAs in Aedes aegypti saliva and salivary glands following chikungunya virus infection

Mosquito saliva facilitates blood feeding through the anti-haemostatic, anti-inflammatory and immunomodulatory properties of its proteins. However, the potential contribution of non-coding RNAs to host manipulation is still poorly understood. We analysed small RNAs from Aedes aegypti saliva and salivary glands and show here that chikungunya virus-infection triggers both the siRNA and piRNA antiviral pathways with limited effects on miRNA expression profiles. Saliva appears enriched in specific miRNA subsets and its miRNA content is well conserved among mosquitoes and ticks, clearly pointing to a non-random sorting and occurrence. Finally, we provide evidence that miRNAs from Ae. aegypti saliva may target human immune and inflammatory pathways, as indicated by prediction analysis and searching for experimentally validated targets of identical human miRNAs. Overall, we believe these observations convincingly support a scenario where both proteins and miRNAs from mosquito saliva are injected into vertebrates during blood feeding and contribute to the complex vector–host–pathogen interactions.

Invading viral DNA triggers dsRNA synthesis by RNA polymerase II to activate antiviral RNA interference in Drosophila.

dsRNA sensing triggers antiviral responses against RNA and DNA viruses in diverse eukaryotes. In Drosophila, Invertebrate iridescent virus 6 (IIV-6), a large DNA virus, triggers production of small interfering RNAs (siRNAs) by the dsRNA sensor Dicer-2. Here, we show that host RNA polymerase II (RNAPII) bidirectionally transcribes specific AT-rich regions of the IIV-6 DNA genome to generate dsRNA. Both replicative and naked IIV-6 genomes trigger production of dsRNA in Drosophila cells, implying direct sensing of invading DNA. Loquacious-PD, a Dicer-2 co-factor essential for the biogenesis of endogenous siRNAs, is dispensable for processing of IIV-6-derived dsRNAs, which suggests that they are distinct. Consistent with this finding, inhibition of the RNAPII co-factor P-TEFb affects the synthesis of endogenous, but not virus-derived, dsRNA. Altogether, our results suggest that a non-canonical RNAPII complex recognizes invading viral DNA to synthesize virus-derived dsRNA, which activates the antiviral siRNA pathway in Drosophila.

Intergenerational Inheritance of Altered Metabolism Phenotypes After Early‐life Stress in Caenorhabditis elegans

Environmental stress experienced during critical periods of animal development has been shown to impact adult phenotypes through changes in behavior or physiology. In humans, undernutrition in utero can lead to obesity and metabolic disorders in adulthood that can be inherited for two generations in the absence of the original stress. However, how particular stresses can result in tissue‐specific changes in target gene expression, and how that gene expression can be inherited, is not well understood. We have established Caenorhabditis elegans nematodes as a model system to investigate the molecular mechanisms that regulate environmental programming of gene expression. C. elegans larvae experiencing early‐life stress (starvation or crowding) may enter the stress resistant, developmentally arrested dauer stage. When environmental conditions improve, larvae will exit dauer and proceed with reproductive development (postdauers, PD). In previous work, we have shown using RNA‐Seq that hermaphrodites that experienced starvation‐induced dauer (PDStv) exhibited significant changes in gene expression compared to continuously fed animals (control, CON), most notably a downregulation of germline‐expressed genes and upregulation of genes with functions in fatty acid metabolism. These changes in gene expression correlated with production of fewer progeny and a decrease in stored intestinal lipids by Oil Red O (ORO) staining in PDStv adults compared to controls. The observed reduction in fecundity in PDStv hermaphrodites was dependent on functional Δ9‐desaturase genes, fat‐5, fat‐6, and fat‐7,as well as the DAF‐12/VDR steroid signaling pathway, indicating a connection between the starvation‐induced metabolism changes and fertility. Interestingly, embryos of PDStv hermaphrodites contained significantly more lipids compared to control embryos, suggesting that PDStv adults prioritize lipid production for reproduction rather than somatic maintenance after dauer exit. We next examined the lipid storage F1 and F2 generation adults using ORO staining, and found that F1 progeny of PDStv adults have significantly increased lipid storage compared to controls. This difference in lipid storage is reset in the F2 generation. Additionally, inheritance of the altered metabolism in F1 adults is dependent upon PRG‐1/PIWI and HRDE‐1/AGO siRNA pathways. In recent work, we are further investigating the connection between metabolism and fecundity of an animal that experienced early‐life stress. We examined the progeny number of PDStv adults that were cultivated on E. coli OP50 supplemented with different fatty acid molecules. We observed that supplementation with oleic acid (OA) resulted in a significant increase in progeny number compared to animals fed only E. coli, while other fatty acids resulted in no change or a decrease in progeny number. Furthermore, the OA‐dependent increase in progeny number required the Δ9‐desaturase FAT‐7, as well as the DAF‐12/VDR steroid signaling pathway, suggesting that OA may be modifying the starvation‐induced phenotypes of PDStv adults. Together, these results suggest that OA may be acting as a signaling molecule to modulate reproductive plasticity through regulation of the DAF‐12/VDR steroid signaling pathway in response to environmental conditions.

The small RNA universe of Capitella teleta

RNAi is an evolutionarily fluid mechanism with dramatically different activities across animal phyla. One major group where there has been little investigation is annelid worms. Here the small RNAs of the polychaete developmental model, Capitella teleta, are profiled across development. As is seen with nearly all animals, nearly 200 hundred microRNAs were found with 58 high confidence novel species. Greater miRNA diversity was associated with later stages consistent with differentiation of tissues. Outside miRNA, a distinct composition of other small RNA pathways was found. Unlike many invertebrates, an endogenous siRNA pathway was not observed, indicating pathway loss relative to basal planarians. No processively generated siRNA‐class RNAs could be found arising from dsRNA precursors. This has significant impact on RNAi technology development for this group of animals. Unlike the apparent absence of siRNAs, a significant population of piRNAs were observed. For many piRNAs phasing and ping pong biogenesis pathways were identified. Interestingly, piRNAs were found to be highly expressed during early development, suggesting a potential role in regulation in metamorphosis. Critically, the configuration of RNAi factors in C. teletais found in other annelids and mollusks, suggesting that similar biology is likely present in the wider clade. This study is the first providing comprehensive analysis of small RNAs in annelids.

RNAi in Piezodorus guildinii (Hemiptera: Pentatomidae): Transcriptome Assembly for the Development of Pest Control Strategies.

Red-banded stink bug Piezodorus guildinii (P. guildinii) has been described as the most damaging stink bug regarding soybean crops, leading to seed injury, low germination percentages, and foliar retention, at low population densities. In recent years, RNA interference (RNAi), a conserved eukaryote silencing mechanism has been explored to develop species-selective pesticides. In this work, we evaluated RNAi in P. guildinii to develop new pest-control strategies. For this, we assembled and annotated a P. guildinii transcriptome from a pool of all developmental stages. Analysis of this transcriptome led to the identification of 56 genes related to the silencing process encompassing siRNA, miRNA, and piRNA pathways. To evaluate the functionality of RNAi machinery, P. guildinii adults were injected with 28 ng/mg of body weight of double stranded RNA (dsRNA) targeting vATPase A. A mortality of 35 and 51.6% was observed after 7 and 14 days, respectively, and a downregulation of vATPase A gene of 84% 72 h post-injection. In addition, Dicer-2 and Argonaute-2 genes, core RNAi proteins, were upregulated 1.8-fold 48 h after injection. These findings showed for the first time that RNAi is functional in P. guildinii and the silencing of essential genes has a significant effect in adult viability. Taken together, the work reported here shows that RNAi could be an interesting approach for the development of red-banded stink bug control strategies.

Genome-wide characterization of miRNA and siRNA pathways in the parasitoid wasp Pteromalus puparum

microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small non-coding RNAs (ncRNAs) that trigger RNA interference (RNAi) in eukaryotic organisms. The biogenesis pathways for these ncRNAs are well established in Drosophila melanogaster, Aedes aegypti, Bombyx mori and other insects, but lacking in hymenopteran species, particularly in parasitoid wasps. Pteromalus puparum is a parasitoid of pupal butterflies. This study identified and analyzed two pathways by interrogating the P. puparum genome. All core genes of the two pathways are present in the genome as a single copy, except for two genes in the siRNA pathway, R2D2 (two copies) and Argonaute-2 (three). Conserved domain analyses showed the protein structures in P. puparum were similar to cognate proteins in other insect species. Phylogenetic analyses of hymenopteran Dicer and Argonaute genes suggested that the siRNA pathway-related genes evolved faster than those in the miRNA pathway. The study found a decelerated evolution rate of P. puparum Dicer-2 with respect to Dicer-1, which was contrary to other hymenopterans. Expression analyses revealed high mRNA levels for all miRNA pathway genes in P. puparum adults and the siRNA related genes were expressed in different patterns. The findings add valuable new knowledge of the miRNA and siRNA pathways and their regulatory actions in parasitoid wasps.

The Small RNA Universe of Capitella teleta.

RNAi is an evolutionarily fluid mechanism with dramatically different activities across animal phyla. One major group where there has been little investigation is annelid worms. Here, the small RNAs of the polychaete developmental model Capitella teleta are profiled across development. As is seen with nearly all animals, nearly 200 microRNAs were found with 58 high-confidence novel species. Greater miRNA diversity was associated with later stages consistent with differentiation of tissues. Outside miRNA, a distinct composition of other small RNA pathways was found. Unlike many invertebrates, an endogenous siRNA pathway was not observed, indicating pathway loss relative to basal planarians. No processively generated siRNA-class RNAs could be found arising from dsRNA precursors. This has a significant impact on RNAi technology development for this group of animals. Unlike the apparent absence of siRNAs, a significant population of piRNAs was observed. For many piRNAs, phasing and ping-pong biogenesis pathways were identified. Interestingly, piRNAs were found to be highly expressed during early development, suggesting a potential role in regulation in metamorphosis. Critically, the configuration of RNAi factors in C. teleta is found in other annelids and mollusks, suggesting that similar biology is likely to be present in the wider clade. This study is the first in providing comprehensive analysis of small RNAs in annelids.

BmNPV p35 Reduces the Accumulation of Virus-Derived siRNAs and Hinders the Function of siRNAs to Facilitate Viral Infection.

Antiviral immunity involves various mechanisms and responses, including the RNA interference (RNAi) pathway. During long-term coevolution, viruses have gained the ability to evade this defense by encoding viral suppressors of RNAi (VSRs). It was reported that p35 of baculovirus can inhibit cellular small interference RNA (siRNA) pathway; however, the molecular mechanisms underlying p35 as a VSR remain largely unclear. Here, we showed that p35 of Bombyx mori nucleopolyhedrovirus (BmNPV) reduces the accumulation of virus-derived siRNAs (vsiRNAs) mapped to a particular region in the viral genome, leading to an increased expression of the essential genes in this region, and revealed that p35 disrupts the function of siRNAs by preventing them from loading into Argonaute-2 (Ago2). This repressive effect on the cellular siRNA pathway enhances the replication of BmNPV. Thus, our findings illustrate for the first time the inhibitory mechanism of a baculovirus VSR and how this effect influences viral infection.

Virus and endogenous viral element-derived small non-coding RNAs and their roles in insect–virus interaction

RNA interference pathways mediated by different types of small non-coding RNAs (siRNAs, miRNAs and piRNAs) are conserved biological responses to exotic stresses, including viral infection. Aside from the well-established siRNA pathway, the miRNA pathway and the piRNA pathway process viral sequences, exogenously or endogenously, into miRNAs and piRNAs, respectively. During the host-virus interaction, viral sequences, including both coding and non-coding sequences, can be integrated as endogenous viral elements (EVEs) and thereby become present within the germline of a non-viral organism. In recent years, significant progress has been made in characterizing the biogenesis and function of viruses and EVEs associated with snRNAs. Overall, the siRNA pathway acts as the primarily antiviral defense against a wide range of exogenous viruses; the miRNA pathways associated with viruses or EVEs function in antiviral response and host gene regulation; EVE derived piRNAs with a ping-pong signature have the potential to limit cognate viral infection.

When Down Is Up: Heterochromatin, Nuclear Organization and X Upregulation.

Organisms with highly differentiated sex chromosomes face an imbalance in X-linked gene dosage. Male Drosophila solve this problem by increasing expression from virtually every gene on their single X chromosome, a process known as dosage compensation. This involves a ribonucleoprotein complex that is recruited to active, X-linked genes to remodel chromatin and increase expression. Interestingly, the male X chromosome is also enriched for several proteins associated with heterochromatin. Furthermore, the polytenized male X is selectively disrupted by the loss of factors involved in repression, silencing, heterochromatin formation or chromatin remodeling. Mutations in many of these factors preferentially reduce male survival or enhance the lethality of mutations that prevent normal recognition of the X chromosome. The involvement of primarily repressive factors in a process that elevates expression has long been puzzling. Interestingly, recent work suggests that the siRNA pathway, often associated with heterochromatin formation and repression, also helps the dosage compensation machinery identify the X chromosome. In light of this finding, we revisit the evidence that links nuclear organization and heterochromatin to regulation of the male X chromosome.

Identification and functional characterization of the siRNA pathway in Taenia crassiceps by silencing Enolase A

A gene silencing procedure on cysticerci of the taeniid cestode Taenia crassiceps is described. This is the first time this technique is reported in this species that is widely used as an animal model for human cysticercosis. Genome database searches were performed in order to find out if relevant genes involved in gene silencing and non-coding RNA processing, Argonaute and Dicer (AGO and Dcr) are present in T. crassiceps. We found three AGO and two Dcr orthologues that were designed TcAGO1, Tc2 and Tc3, as well as TcDcr1 and TcDcr2. In order to elucidate the evolutionary relationships of T. crassiceps TcAGO and TcDcr genes, separate phylogenetic analyses were carried out for each, including AGO and Dcr orthologues of other 20 platyhelminthes. Our findings showed a close phylogenetic relationship of TcAGO and TcDcr with those previously described for Echinococcus spp. Our RT-PCR studies demonstrated expression of all TcAGO and TcDcr orthologues. Our results show that the gene silencing machinery in T. crassiceps is functionally active by inducing silencing of TcEnoA (∼90%). These results clearly show that gene silencing using siRNAs can be used as a molecular methodology to study gene function in taeniid cestodes.

Molecular Characterizations and Functional Analyses of LmR2D2 in the Locusta migratoria siRNA Pathway.

Simple SummaryLocusta migratoria is a serious agricultural pest all over the world, which devastates agriculture and challenges food security and livelihoods. Recently, RNA interference (RNAi) technology has emerged as a novel strategy for managing insect pests. However, different insect species show a significant variation in RNAi efficiency. Therefore, it is necessary to better understand the RNAi mechanism. In this study, we identified an LmR2D2 gene in the L. migratoria transcriptome. After the expression of LmR2D2 was suppressed by RNAi, we found a significantly diminished RNAi efficiency against a marker gene in L. migratoria. Our binding experiments further demonstrated that the LmR2D2 protein can bind double-stranded RNA (dsRNA) in vitro. On the basis of these results, we conclude that LmR2D2 is involved in the L. migratoria siRNA pathway.Small interfering RNAs (siRNAs) are non-coding RNAs with a length of 21~23 nucleotides (nt) and present in almost all eukaryotes. The formation of siRNA is a highly conserved post-transcriptional gene-silencing mechanism mediated by key proteins, including Dicer2, Argonaute2 (Ago2) and R2D2. R2D2 has been identified as a double-stranded RNA (dsRNA)-binding protein and reported as an integral component of the siRNA pathway in Drosophila. However, the involvement of R2D2 in the siRNA pathway of Locusta migratoria is still unknown. In the present study, we identified an LmR2D2 gene from the transcriptome of L. migratoria. It consists of a 954-bp open reading frame that encodes a protein of 318 amino acid residues. Further sequence analysis revealed that LmR2D2 possesses two tandem dsRNA-binding domains (dsRBD) at the N-terminus. Analysis of the developmental expression profile of LmR2D2 indicated that its transcript level was stable in third-instar nymphs of L. migratoria, whereas the tissue-dependent expression profile exhibited high levels of expression of LmR2D2 in the testis and ovary. When LmR2D2 was silenced by RNAi, the RNAi efficiency against Lmβ-tubulin as a marker gene was significantly diminished, as indicated by the 37.7% increased Lmβ-tubulin transcript level. Additionally, the prokaryotic expression system was used to obtain the LmR2D2 supernatant protein. By incubating the LmR2D2 protein with biotin-dsRNA, we found that LmR2D2 can bind to dsRNA in vitro, which supports our conclusion that LmR2D2 plays an essential role in the siRNA pathway of L. migratoria.

Regulation of RNA Interference Pathways in the Insect Vector Laodelphax striatellus by Viral Proteins of Rice Stripe Virus.

RNA interference (RNAi), especially the small interfering RNA (siRNA) and microRNA (miRNA) pathways, plays an important role in defending against viruses in plants and insects. However, how insect-transmitted phytoviruses regulate the RNAi-mediated antiviral response in vector insects has barely been uncovered. In this study, we explored the interaction between rice stripe virus (RSV) and the miRNA and siRNA pathways of the small brown planthopper, which is a vector insect. The transcript and protein levels of key genes in the two RNAi pathways did not change during the RSV infection process. When the expression of insect Ago1, Ago2, or Translin was silenced by the injection of double-stranded RNAs targeting these genes, viral replication was promoted with Ago2 silencing but inhibited with Translin silencing. Protein-protein binding assays showed that viral NS2 and RNA-dependent RNA polymerase interacted with insect Ago2 and Translin, respectively. When NS2 was knocked down, the transcript level of Ago2 increased and viral replication was inhibited. Therefore, viral NS2 behaved like an siRNA suppressor in vector insects. This protein-binding regulation of insect RNAi systems reflects a complicated and diverse coevolution of viruses with their vector insects.

NAP1L1 Functions as a Tumor Promoter via Recruiting Hepatoma-Derived Growth Factor/c-Jun Signal in Hepatocellular Carcinoma.

NAP1L1 has been reported to be significantly involved in the carcinogenesis of hepatocellular carcinoma (HCC). Yet, its detailed molecular basis is still to be determined. Based on the analysis of The Cancer Genome Atlas (TCGA) database, NAP1L1 mRNA was found to be upregulated and predicted the poor prognosis initially. Subsequently, consistent with the prediction, the upregulated expression of NAP1L1 mRNA and protein levels was confirmed by quantitative polymerase chain reaction (qPCR), Western blot, and immunohistochemistry assays. Upregulated NAP1L1 protein positively promoted the disease progression and poor prognosis of HCC. In addition, NAP1L1 protein expression was considered as an independent prognostic factor in HCC. Inhibition of NAP1L1 expression by siRNA or shRNA pathway significantly reduced the cell proliferation and cell cycle transformation in vitro and in vivo. Mechanism analysis first showed that the function of NAP1L1 was to recruit hepatoma-derived growth factor (HDGF), an oncogene candidate widely documented in tumors. Furthermore, the latter interacted with c-Jun, a key oncogenic transcription factor that can induce the expression of cell cycle factors and thus stimulate the cell growth in HCC. Finally, transfecting HDGF or c-Jun could reverse the suppressive effects on HCC growth in NAP1L1-suppressed HCC cells. Our data indicate that NAP1L1 is a potential oncogene and acts via recruiting HDGF/c-Jun in HCC.

An Aedes aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections.

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

Gaining Acceptance of Novel Plant Breeding Technologies.

Ensuring the sustainability of agriculture under climate change has led to a surge in alternative strategies for crop improvement. Advances in integrated crop breeding, social acceptance, and farm-level adoption are crucial to address future challenges to food security. Societal acceptance can be slow when consumers do not see the need for innovation or immediate benefits. We consider how best to address the issue of social licence and harmonised governance for novel gene technologies in plant breeding. In addition, we highlight optimised breeding strategies that will enable long-term genetic gains to be achieved. Promoted by harmonised global policy change, innovative plant breeding can realise high and sustainable productivity together with enhanced nutritional traits.

Micro RNA-induced gene silencing strategy for the delivery of siRNAs targeting Meloidogyne incognita in a model plant Nicotiana benthamiana.

Occurrence of multiple biotic stresses on crop plants result in drastic yield losses which may have severe impact on the food security. It is a challenge to design strategies for simultaneous management of these multiple stresses. Hence, establishment of innovative approaches that aid in their management is critical. Here, we have introgressed a micro RNA-induced gene silencing (MIGS) based combinatorial gene construct containing seven target gene sequences of cotton leaf curl disease (CLCuD), cotton leaf hopper (Amrasca biguttula biguttula), cotton whitefly (Bemisia tabaci) and root-knot nematode (Meloidogyne incognita). Stable transgenic lines of Nicotiana benthamiana were generated with the T-DNA harboring Arabidopsis miR173 target site fused to fragments of Sec23 and ecdysone receptor (EcR) genes of cotton leaf hopper and cotton whitefly. It also contained C2/replication associated protein (C2/Rep) and C4 (movement protein) along with βC1 gene of betasatellite to target CLCuD, and two FMRFamide-like peptide (FLP) genes, Mi-flp14 and Mi-flp18 of M. incognita. These transgenic plants were assessed for the amenability of MIGS approach for pest control by efficacy evaluation against M. incognita. Results showed successful production of small interfering RNA (siRNA) through the tasiRNA (trans-acting siRNA) pathway in the transgenic plants corresponding to Mi-flp18 gene. Furthermore, we observed reduced Mi-flp14 and Mi-flp18 transcripts (up to 2.37 ± 0.12-fold) in females extracted from transgenic plants. The average number of galls, total endoparasites, egg masses and number of eggs per egg mass reduced were in the range 27-62%, 39-70%, 38-65% and 34-49%, respectively. More importantly, MIGS transgenic plants showed 80% reduction in the nematode multiplication factor (MF). This study demonstrates successful validation of the MIGS approach in the model plant, N. benthamiana for efficacy against M. incognita, as a prelude to translation to cotton. © 2021 Society of Chemical Industry.

A Systems Biology Approach to Identify Essential Epigenetic Regulators for Specific Biological Processes in Plants.

Upon sensing developmental or environmental cues, epigenetic regulators transform the chromatin landscape of a network of genes to modulate their expression and dictate adequate cellular and organismal responses. Knowledge of the specific biological processes and genomic loci controlled by each epigenetic regulator will greatly advance our understanding of epigenetic regulation in plants. To facilitate hypothesis generation and testing in this domain, we present EpiNet, an extensive gene regulatory network (GRN) featuring epigenetic regulators. EpiNet was enabled by (i) curated knowledge of epigenetic regulators involved in DNA methylation, histone modification, chromatin remodeling, and siRNA pathways; and (ii) a machine-learning network inference approach powered by a wealth of public transcriptome datasets. We applied GENIE3, a machine-learning network inference approach, to mine public Arabidopsis transcriptomes and construct tissue-specific GRNs with both epigenetic regulators and transcription factors as predictors. The resultant GRNs, named EpiNet, can now be intersected with individual transcriptomic studies on biological processes of interest to identify the most influential epigenetic regulators, as well as predicted gene targets of the epigenetic regulators. We demonstrate the validity of this approach using case studies of shoot and root apical meristem development.

The Interplay Between Viruses and RNAi Pathways in Insects.

As an overarching immune mechanism, RNA interference (RNAi) displays pathogen specificity and memory via different pathways. The small interfering RNA (siRNA) pathway is the primary antiviral defense mechanism against RNA viruses of insects and plays a lesser role in defense against DNA viruses. Reflecting the pivotal role of the siRNA pathway in virus selection, different virus families have independently evolved unique strategies to counter this host response, including protein-mediated, decoy RNA-based, and microRNA-based strategies. In this review, we outline the interplay between insect viruses and the different pathways of the RNAi antiviral response; describe practical application of these interactions for improved expression systems and for pest and disease management; and highlight research avenues for advancement of the field.

Knockdown of Target Genes by siRNA In Vitro.

RNA interference (RNAi) is a cellular process involved in the silencing of genes, which makes RNAi important for observing and understanding the function of specific gene products. Short interfering RNA (siRNA) pathway is a RNAi pathway, where exogenous double stranded RNA is introduced to the cell and cleaved by an endoribonuclease, Dicer, to form siRNA, which interacts with a protein complex to scan mRNAs to bind to its complementary sequence. The binding of the siRNA to its complementary mRNA, the mRNA is cleaved and degraded by the cell, significantly reducing the levels of the target protein product. The discovery of this mechanism made it a powerful tool to use as a technique for therapeutics, agricultural biology, and cellular and molecular biology.