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Argonaute proteins are key constituents of small RNA-guided regulatory pathways. In crustaceans, members of the AGO subfamily of Argonaute proteins that play vital roles in immune defense are well studied, while proteins of the PIWI subfamily are less established. PmAgo4 of the black tiger shrimp, Penaeus monodon, though phylogenetically clustered with the AGO subfamily, has distinctive roles of the PIWI subfamily in safeguarding the genome from transposon invasion and controlling germ cell development. This study explored a molecular mechanism by which PmAgo4 regulates transposon expression in the shrimp germline. PmAgo4-associated small RNAs were co-immunoprecipitated from shrimp testis lysate using a PmAgo4-specific polyclonal antibody. RNA-seq revealed a majority of 26-27 nt long small RNAs in the PmAgo4-IP fraction suggesting that PmAgo4 is predominantly associated with piRNAs. Mapping of these piRNAs on nucleotide sequences of two gypsy and a mariner-like transposons of P. monodon suggested that most piRNAs were originated from the antisense strand of transposons. Suppression of PmAgo4 expression by a specific dsRNA elevated the expression levels of the three transposons while decreasing the levels of transposon-related piRNAs. Taken together, these results imply that PmAgo4 exerts its suppressive function on transposons by controlling the biogenesis of transposon-related piRNAs and thus, provides a defense mechanism against transposon invasion in shrimp germline cells., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases., Methods: Two distinct approaches (immunoprecipitation/mass spectrometry-based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples., Results: Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments., Conclusions: AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder., Classification of Evidence: This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Objectives: The presence of myositis-specific antibodies (MSA), was recently reported in healthy individuals, cancer patients without myopathy and paraneoplastic rheumatic syndromes. We sought to analyze the frequency of MSA, myositis-associated antibodies (MAA) and autoantibodies related to systemic autoimmune rheumatic diseases (SARD) in breast cancer patients., Methods: One hundred fifty-two breast cancer patients were enrolled in a cross-sectional study. Clinical information was collected, and autoantibodies tested by immunoprecipitation of an 35 S-methionine-labeled K562 cell extract, enzyme-linked immunosorbent assay (ELISA) and Western blot when indicated. All statistical tests were performed using the software statistical package for the social science (SPSS) ver. 19.0 (IBM Inc., NYSE, USA)., Results: Autoantibodies associated with SARD: anti-52 kD ribonucleoprotein/tripartite motif-containing 21 (anti-Ro52/TRIM21) was found in 5.9% (9/152), anti-Sjögren syndrome-related antigen A/60 kD ribonucleoprotein antibody (anti-SSA/Ro60) in 3.9% (6/152) and anti-Su antigen/Argonaute 2 antibody (anti-Su/Ago2) in 2.6% (4/152). Meanwhile, anti-transcription intermediary factor-1γ (anti-TIF-1γ, p155/140) antibody was positive in 2 cases and anti-polymyositis/scleroderma antibody was detected in one case. As a whole, 14.47% (22/152) of breast cancer patients showed autoantibodies associated with SARD. These specific autoantibodies were not associated with the presence of rheumatic diseases except one rheumatoid arthritis patient positive for anti-Ro52/TRIM21., Conclusions: Autoantibodies to TIF-1γ were found in two patients with breast cancer without dermatomyositis (DM). More common specificities were autoantibodies anti-SSA/Ro60, anti-Ro52/TRIM21 and anti-Su/Ago2. More studies are needed in order to establish the biological meaning of the presence of SARD-associated autoantibodies in breast cancer.

During infection, RNA viruses can produce two types of virus-derived small RNAs (vsRNAs), small interfering RNA (siRNA) and microRNA (miRNA), that play a key role in RNA silencing-mediated antiviral mechanisms in various hosts by associating with different Argonaute (Ago) proteins. Ago1 has been widely identified as an essential part of the miRNA pathway, while Ago2 is required for the siRNA pathway. Thus, analysis of the interaction between vsRNAs and Ago proteins can provide a clue about which pathway the vsRNA may be involved in. In this study, using rice stripe virus (RSV)-small brown planthoppers (Laodelphax striatellus, Fallen) as an infection model, the interactions of eight vsRNAs derived from four viral genomic RNA fragments and Ago1 or Ago2 were detected via the RNA immunoprecipitation (RIP) method. vsRNA4-1 and vsRNA4-2 derived from RSV RNA4 were significantly enriched in Ago1-immunoprecipitated complexes, whereas vsRNA2-1 and vsRNA3-2 seemed enriched in Ago2-immunoprecipitated complexes. vsRNA1-2 and vsRNA2-2 were detected in both of the two Ago-immunoprecipitated complexes. In contrast, vsRNA1-1 and vsRNA3-1 did not accumulate in either Ago1- or Ago2-immunoprecipitated complexes, indicating that regulatory pathways other than miRNA or siRNA pathways might be employed. In addition, two conserved L. striatellus miRNAs were analysed via the RIP method. Both miRNAs accumulated in Ago1-immunoprecipitated complexes, which was consistent with previous studies, suggesting that our experimental system can be widely used. In conclusion, our study provides an accurate and convenient detection system to determine the potential pathway of vsRNAs, and this method may also be suitable for studying other sRNAs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Argonaute protein (AGO2) bound circulating cell-free miRNAs (ccf-miRs), in the recent years, has attracted great attention due to their differential abundance in biological fluids. In the present work, a selective and technically uncomplicated quantum dot (QD) nanoconjugate has been fabricated combining the specific affinity of the antibody and fluorescent property of QDs for the precise immuno-detection of AGO2-bound ccf-miRs in plasma samples. The electrophoretic mobility assay confirmed the conjugation of antibody with QDs. The detection methodology involves a highly specific antigen-antibody reaction between the AGO2 proteins of miRNA-induced silencing complex and the anti-AGO2 antibody conjugated with QDs. The recognition efficiency of QD-Ab nanoconjugates was analysed using flow cytometry and fluorometry. The flow cytometry results demonstrated a significant change in the fluorescence intensity of the prepared nanoconjugates upon capture of ccf-miRs in the plasma samples with respect to the samples devoid of any miRNAs. Fluorometry measurements exhibited corroboration with the flow cytometry results indicating the selectivity and reproducibility of the developed method. Current research highlights the translational significance of the methodology as a novel flow cytometry based immunoassay for detection of differentially expressed AGO2-bound miRNAs in clinical and field settings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

RNAi (RNA interference) is an important defense response against virus infection in plants. The core machinery of the RNAi pathway in plants include DCL (Dicer Like), AGO (Argonaute) and RdRp (RNA dependent RNA polymerase). Although involvement of these RNAi components in virus infection responses was demonstrated in Arabidopsis thaliana , their contribution to antiviral immunity in Nicotiana benthamiana , a model plant for plant-pathogen interaction studies, is not well understood. In this study, we investigated the role of N. benthamiana NbAGO2 gene against TMV (Tomato mosaic virus) infection. Silencing of NbAGO2 by transient expression of an hpRNA construct recovered GFP (Green fluorescent protein) expression in GFP -silenced plant, demonstrating that NbAGO2 participated in RNAi process in N. benthamiana . Expression of NbAGO2 was transcriptionally induced by both MeSA (Methylsalicylate acid) treatment and TMV infection. Down-regulation of NbAGO2 gene by amiR- NbAGO2 transient expression compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2 , reduced TMV infection. Our study provides evidence for the antiviral role of NbAGO2 against a Tobamovirus family virus TMV in N. benthamiana , and SA (Salicylic acid) mediates this by induction of NbAGO2 expression upon TMV infection. Our data also highlighted that miR403a was involved in TMV defense by regulation of target NbAGO2 gene in N. Benthamiana .

RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green, and blue imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis ( Arabidopsis thaliana ) plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent nonadditive, anti-TCV effectors, whereas AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses., (Copyright © 2019. Published by Elsevier Ltd.)

Immune responses in insects are differentially triggered depending on the infection route used by the pathogen. In most studies involving Drosophila melanogaster and viruses, infection is done by injection, while oral infection, which is probably the most common route of viral entry in nature, remains unexplored. Here, we orally infected adults and larvae from wild-type and RNA interference (RNAi) mutant flies with different RNA viruses. We found that, in contrast with what is observed following virus injection, oral infections initiated at larval or adult stages are cleared in adult flies. Virus elimination occurred despite a larger infectious dose than for injected flies and evidence of viral replication. RNAi mutant flies suffered greater mortality relative to wild-type flies following oral infection, but they also eliminated the virus, implying that RNAi is not essential for viral clearance and that other immune mechanisms act during oral infections. We further showed that information of infection by RNA viruses acquired orally leaves a trace under a DNA form, which confers protection against future reinfection by the same virus. Together, this work presents evidence of clearance and immune priming for RNA viruses in insects and challenges the current view of antiviral immunity in insects.

Viruses employ intricate means to evade host innate immune systems. In this issue of Cell Host & Microbe, Nayak et al. (2018) demonstrate that the cricket paralysis virus (CrPV) protein CrPV-1A blocks host defense through a dual mechanism: directly inhibiting Argonaute2 (Ago2) and simultaneously targeting Ago2 for proteasomal degradation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a well-known virus in the Baculoviridae family. Presence of the p35 gene in the AcMNPV genome as a suppressor of the short interfering RNA (siRNA) pathway is a strong reason for the importance of the siRNA pathway in the host cellular defense. Given that, here we explored the roles of Dicer-2 (Dcr2) and Argonaute 2 (Ago2) genes, key factors in the siRNA pathway in response to AcMNPV infection in Spodoptera frugiperda Sf9 cells. The results showed that the transcript levels of Dcr2 and Ago2 increased in response to AcMNPV infection particularly over 16 h post infection suggesting induction of the siRNA pathway. Reductions in the expression levels of Dcr2 and Ago2 by using specific dsRNAs in Sf9 cells modestly enhanced production of viral genomic DNA which indicated their role in the host antiviral defense. Using deep sequencing, our previous study showed a large number of small reads (siRNAs of ∼20 nucleotides) from AcMNPV-infected Sf9 cells that were mapped to some of the viral genes (hot spots). Down-regulation of Dcr2 in Sf9 cells resulted in enhanced expression levels of the selected virus hotspot genes (i.e. ORF-9 and ORF-148), while the transcript levels of virus cold spots (i.e. ORF-18 and ORF-25) with no or few siRNAs mapped to them did not change. Overexpression of AcMNPV p35 as a suppressor of RNAi and anti-apoptosis gene in Sf9 cells increased virus replication. Also, replication of mutant AcMNPV lacking the p35 gene was significantly increased in Sf9 cells with reduced transcript levels of Dcr2 and Ago2, highlighting the antiviral role of the siRNA pathway in Sf9 cells. Together, our results demonstrate that Dcr2 and Ago2 genes contribute in efficient antiviral response of Sf9 cells towards AcMNPV, and in turn, the AcMNPV p35 suppresses the siRNA pathway, besides being an antiapoptotic protein., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The control of viral infections in insects is a current issue of major concern and RNA interference (RNAi) is considered the main antiviral immune response in this group of animals. Here we demonstrate that overexpression of key RNAi factors can help to protect insect cells against viral infections. In particular, we show that overexpression of Dicer2 and Argonaute2 in lepidopteran cells leads to improved defense against the acute infection of the Cricket Paralysis Virus (CrPV). We also demonstrate an important role of RNAi in the control of persistent viral infections, as the one caused by the Macula-like Latent Virus (MLV). Specifically, a direct interaction between Argonaute2 and virus-specific small RNAs is shown. Yet, while knocking down Dicer2 and Argonaute2 resulted in higher transcript levels of the persistently infecting MLV in the lepidopteran cells under investigation, overexpression of these proteins could not further reduce these levels. Taken together, our data provide deep insight into the RNAi-based interactions between insects and their viruses. In addition, our results suggest the potential use of an RNAi gain-of-function approach as an alternative strategy to obtain reduced viral-induced mortality in Lepidoptera, an insect order that encompasses multiple species of relevant economic value.

The microRNA (miRNA) biogenesis pathway is regulated by specific proteins and enzymes, including Dicer, Drosha, DGCR8, Exportin 5 and the Argonaute (AGO) family. In this study, we investigated the AGO family, which is the primary component of RISC (RNA-induced silencing complex) and directly binds to microRNA. We examined the association of polymorphisms in AGO family genes with AGO expression and with the development and prognosis of autoimmune thyroid diseases. We genotyped AGO1 rs636832A/G, AGO2 rs7005286C/T, AGO2 rs11166985A/G and AGO2 rs2292779C/G polymorphisms in 184 Graves' disease (GD) patients, 195 Hashimoto's disease (HD) patients and 122 healthy volunteers using the polymerase chain reaction-restriction fragment length polymorphism method. We also examined the expression of AGO1 and AGO2 mRNAs in peripheral blood mononuclear cells (PBMC) obtained from 52 GD patients, 41 HD patients, and 25 healthy volunteers using quantitative RT-PCR methods. The G allele of AGO1 rs636832 and the A allele of AGO2 rs11166985 polymorphisms were significantly more frequent in GD patients than in healthy controls. The A allele of AGO2 rs11166985 was also significantly more frequent in intractable GD patients than in controls. The C carrier (CC + CG genotypes) and C allele of AGO2 rs2292779 polymorphism were significantly more frequent in intractable GD patients than in patients with GD in remission. Expression of AGO1 mRNA in PBMC was significantly higher in AITD patient than in controls, and that of AGO2 mRNA in PBMC was significantly higher in intractable GD patients than in patients with GD in remission. Furthermore, the expression levels of both the AGO1 and AGO2 genes were significantly correlated with the proportions of Th17 cells in PBMC. In conclusion, the polymorphisms of the AGO1 and AGO2 genes, the expression levels of which correlated with the proportion of Th17 cells, were associated with the development and prognosis of GD. The AGO2 rs2292779 C carrier and C allele were associated with the intractability of GD.

MicroRNAs (miRNAs) are small non-coding RNAs that trigger post-transcriptional gene silencing. These RNAs need to be associated with the Argonaute proteins to be functional. This assembly begins with loading of a miRNA duplex, followed by the ejection of one of the strands (passenger). The remaining strand (guide) together with the Argonaute protein forms a ribonucleoprotein effector complex (the RNA-induced silencing complex, RISC). Mutation on the Argonaute protein, if affecting either step of the RISC assembly, impacts the function of miRNAs. Therefore, any observation of decreased miRNA level of mutants will provide insights into the role of those amino acid residues in the mechanical function of the Argonaute protein. In this chapter, we introduce a method to relatively quantify a specific miRNA co-immunoprecipitated with wild type and mutant Argonaute proteins from HEK293T cells, using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Spiking a synthetic exogenous miRNA as an internal control with RNA extraction prior to cDNA synthesis will normalize the C t values obtained from the qRT-PCR assays and enable us to quantify the relative level of Argonaute-bound miRNA.

Complex immunoprecipitation (Co-IP) is a powerful technique for precipitating an intact protein complex out of solution and cell lysates using an antibody that specifically binds to a particular protein in a large complex of proteins. Mass spectrometry (MS) is used to identify, sequence, and quantify proteins. RNA-induced silencing complexes (RISCs), Ago2 centered protein assemblies, are essential for miRNA mediated RNA decay and gene expression regulation; however, the complete list of RISCs is unknown. Here we describe methods used to combine IP and MS to identify new components of RISCs.

Plants have evolved a variety of defense mechanisms to tackle virus attack. Endogenous plant proteins can function as virus suppressors. Different types of proteins mediate defense responses against plant viruses. Pathogenesis-related (PR) proteins are activated upon pathogen infections or in different stress situations and their production is one of many components in plant defense. Ribosome-inactivating proteins (RIPs) suppress translation by enzymatically damaging ribosomes and they have been found to have antiviral activity. RNA-binding proteins (RBPs) bind to target RNAs via specialized RNA-binding domain and can directly or indirectly function in plant defense system against RNA viruses. Proteins involved in silencing machinery, namely Dicer-like (DCL) proteins, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDRs) confer innate antiviral defense in plants as they are able to degrade foreign RNA of viral origin. This review aims to provide a comprehensive and up-to-date picture of plant proteins participating in antiviral defense. As a result we discuss proteins conferring plant antiviral resistance and their potential future applications in different fields of life including agriculture and medicine., Competing Interests: The authors declare no conflict of interest.

P-element-induced wimpy testes (Piwi) proteins are known for suppressing retrotransposon activation in the mammalian germline. However, whether Piwi protein or Piwi-dependent functions occur in the mammalian soma is unclear. Contrary to germline-restricted expression, we observed that Piwi-like Miwi2 mRNA is indeed expressed in epithelial cells of the lung in adult mice and that it is induced during pneumonia. Further investigation revealed that MIWI2 protein localized to the cytoplasm of a discrete population of multiciliated airway epithelial cells. Isolation and next-generation sequencing of MIWI2-positive multiciliated cells revealed that they are phenotypically distinct from neighboring MIWI2-negative multiciliated cells. Mice lacking MIWI2 exhibited an altered balance of airway epithelial cells, demonstrating fewer multiciliated cells and an increase in club cells. During pneumococcal pneumonia, Miwi2-deficient mice exhibited increased expression of inflammatory mediators and increased immune cell recruitment, leading to enhanced bacterial clearance. Taken together, our data delineate MIWI2-dependent functions outside of the germline and demonstrate the presence of distinct subsets of airway multiciliated cells that can be discriminated by MIWI2 expression. By demonstrating roles for MIWI2 in airway cell identity and pulmonary innate immunity, these studies elucidate unanticipated physiological functions for Piwi proteins in somatic tissues.

Ethanol exposure at the time of burn injury is a major contributor to post-burn pathogenesis. Many of the adverse effects associated with ethanol and burn injury are linked to an impaired intestinal barrier. The combined insult causes intestinal inflammation, resulting in tissue damage, altered tight junction expression, and increased intestinal permeability. MicroRNAs play a critical role in maintaining intestinal homeostasis including intestinal inflammation and barrier function. Specifically, miR-150 regulates inflammatory mediators which can contribute to gut barrier disruption. The present study examined whether ethanol and burn injury alter expression of microRNA processing enzymes (Drosha, Dicer, and Argonaute-2) and miR-150 in the small intestine. Male mice were gavaged with ethanol (~2.9g/kg) 4h prior to receiving a ~12.5% total body surface area full thickness burn. One or three days after injury, mice were euthanized and small intestinal epithelial cells (IECs) were isolated and analyzed for expression of microRNA biogenesis components and miR-150. Dicer mRNA and protein levels were not changed following the combined insult. Drosha and Argonaute-2 mRNA and protein levels were significantly reduced in IECs one day after injury; which accompanied reduced miR-150 expression. To further determine the role of miR-150 in intestinal inflammation, young adult mouse colonocytes were transfected with a miR-150 plasmid and stimulated with LPS (100ng/ml). miR-150 overexpression significantly reduced IL-6 and KC protein levels compared to vector control cells challenged with LPS. These results suggest that altered microRNA biogenesis and associated decrease in miR-150 likely contribute to increased intestinal inflammation following ethanol and burn injury., (Copyright © 2017 Elsevier B.V. All rights reserved.)

P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4 + T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNA Arg (UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4 + T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements., (Copyright © 2017 American Society for Microbiology.)

In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. Here, we explore the variation in the ability to restrain an alien TE in different Drosophila strains. The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster. Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements. The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which we have observed in one particular transposon-resistant R strain.

Prokaryotes and eukaryotes evolved relatively similar RNA-based molecular mechanisms to fight potentially deleterious nucleic acids coming from phages, transposons, or viruses. Short RNAs guide effector complexes toward their targets to be silenced or eliminated. These short immunity RNAs are transcribed from clustered loci. Unexpectedly and strikingly, bacterial and eukaryotic immunity RNA clusters share substantial functional and mechanistic resemblances in fighting nucleic acid intruders., (© 2017 Felden and Paillard; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer., (© 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The mouse testis has served as a popular model system to study a wide range of biological processes, including germ cell development, meiosis, epigenetic changes of chromatin, transposon silencing, and small RNA-mediated epigenetic modifications. PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are almost exclusively expressed in animal gonads. They repress transposons by forming effector complexes with PIWI proteins to maintain genome integrity of the germline. Here we describe detailed procedures of how to produce monoclonal antibodies against a mouse nuclear PIWI protein, MIWI2, which functions in de novo DNA methylation of target transposon loci. We then describe how to use the antibodies to isolate associated complexes and to detect MIWI2 immunohistochemically.

MicroRNAs (miRNAs) are endogenous, small (18-24nt), non-coding RNAs that regulate gene expression. Among miRNAs, those bound to the AGO2 protein are the functionally active fraction which mediates the cell regulatory processes and regulate messages exchanged by cells. Several methods have been developed to purify this fraction of microRNAs, such as immunoprecipitation and immunoprecipitation-derived techniques. However, all these techniques are generally recognized as technically complicated and time consuming. Here, a new bio-functional surface for the specific capture of AGO2-bound microRNAs is proposed. Starting from a silicon oxide surface, a protein A layer was covalently bound via epoxy chemistry to orient specific anti-AGO2 antibodies on the surface. The anti-AGO2 antibodies captured the AGO2 protein present in cell lysate and in human plasma. The AGO2-bound microRNAs were then released by enzymatic digestion and detected via RT-qPCR. Control surfaces were also prepared and tested. Every step in the preparation of the bio-functional surfaces was fully characterized from the chemical, morphological and functional point of view. The resulting bio-functional surface is able to specifically capture the AGO2-bound miRNAs from biologically-relevant samples, such as cell lysate and human plasma. These samples contain different proportions of AGO2-bound microRNAs, as reliably detected with the immunocapture method here proposed. This work opens new perspectives for a simple and faster method to isolate not only AGO2-bound microRNAs, but also the multiprotein complex containing AGO2 and miRNAs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The high frequency of interactions amongst closely related individuals in social insect colonies enhances pathogen transmission. Group-mediated behavior supporting immune defenses tends to decrease selection acting on immune genes. Along with low effective population sizes this might result in relaxed constraint and rapid evolution of immune system genes. Here, we show that antiviral siRNA genes show high rates of molecular evolution with argonaute 2, armitage and maelstrom evolving faster in social bumblebees compared to their socially parasitic cuckoo bumblebees that lack a worker caste. RNAi genes show frequent positive selection at the codon level additionally supported by the occurrence of parallel evolution. Their evolutionary rate is linked to their pathway specific position with genes directly interacting with viruses showing the highest rates of molecular evolution. We suggest that higher pathogen load in social insects indeed drives the molecular evolution of immune genes including antiviral siRNA, if not compensated by behavior., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus-insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 10(14) copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses.

The present study aimed to analyze the contribution of Nicotiana benthamiana ARGONAUTE2 (NbAGO2) to its antiviral response against different viruses. For this purpose, dsRNA hairpin technology was used to reduce NbAGO2 expression in transgenic plants as verified with RT-PCR. This reduction was specific because the expression of other NbAGOs was not affected, and did not cause obvious developmental defects under normal growth conditions. Inoculation of transgenic plants with an otherwise silencing-sensitive GFP-expressing Tomato bushy stunt virus (TBSV) variant resulted in high GFP accumulation because antiviral silencing was compromised. These transgenic plants also exhibited accelerated spread and/or enhanced susceptibility and symptoms for TBSV mutants defective for P19 or coat protein expression, other tombusviruses, Tobacco mosaic virus, and Potato virus X; but not noticeably for Foxtail mosaic virus. These findings support the notion that NbAGO2 in N. benthamiana can contribute to antiviral defense at different levels., (Copyright © 2015. Published by Elsevier Inc.)

Background: Schistosomiasis, caused by schistosomes, is one of the most prevalent and serious parasitic diseases in tropical and subtropical countries. This pathogen has a complex life cycle and harbors a unique repertoire of genes expressed at different life-stages. Understanding the gene regulation of schistosomes will contribute to identification of novel drug targets and vaccine candidates. Some conserved and novel microRNAs (miRNAs) have been identified in schistosomes as key transcriptional and post-transcriptional regulators in the past few years; however, little is known about their specific targets., Methods: High-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation (HITS-CLIP) was used to covalently crosslink native Argonaute protein-RNA complexes in Schistosoma japonicum. An antibody against S.japonicum Argonaute proteins, was generated and used for immunoprecipitation of the crosslinked SjAgo-RNA complex from soluble adult worm extract. Small RNAs, including miRNAs and their target mRNAs associated with the native SjAgo in adult parasites, were enriched and extracted for library construction., Results: High-throughput sequencing produced a total of ~7.4 million high-quality reads, of which approximately 45.07 % were composed of 769 miRNAs and 35.54 % were composed of 11,854 mRNAs target sites. Further bioinformatics analysis identified 43 conserved known miRNAs and 256 novel miRNAs in the SjAgo-associated small RNA population. An average of approximately 15 target sites were predicted for each miRNA. Moreover, a positive rate of 50 % has been achieved in a small-scale verification test of the putative target sites of miRNA1., Conclusion: In this study, we isolated and identified small RNAs including miRNAs and their targets associated with the S. japonicum Argonaute proteins, by the HITS-CLIP method combined with bioinformatics and biologic experimental analysis. These data reveal a genome-wide miRNA-mRNA interaction map in S. japonicum in vivo, which will help us understand the complex gene regulatory network in this pathogen and thereby facilitate the development of novel drug approaches against schistosomiasis.

Profiling small RNAs in soybean Williams 82 (rsv), susceptible to Soybean mosaic virus (SMV, the genus Potyvirus, family Potyviridae) strains G2 and G7, and soybean PI96983 (Rsv1), resistant to G2 but susceptible to G7, identified the microRNA miR168 that was highly overexpressed only in G7-infected PI96983 showing a lethal systemic hypersensitive response (LSHR). Overexpression of miR168 was in parallel with the high-level expression of AGO1 mRNA, high-level accumulation of miR168-mediated AGO1 mRNA cleavage products but with severely repressed AGO1 protein. In contrast, AGO1 mRNA, degradation products and protein remained without significant changes in G2- and G7-infected Williams 82. Moreover, knock-down of SGS3, an essential component in RNA silencing, suppressed AGO1 siRNA, partially recovered repressed AGO1 protein, and alleviated LSHR severity in G7-infected Rsv1 soybean. These results suggest that both miRNA and siRNA pathways are involved in G7 infection of Rsv1 soybean, and LSHR is associated with breakdown of AGO1 homeostasis., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

An anti-inflammatory phenotype with pronounced immunosuppression develops during sepsis, during which time neutrophils and monocytes/macrophages limit their Toll-like receptor 4 responses to bacterial lipopolysaccharide (LPS/endotoxin). We previously reported that during this endotoxin-tolerant state, distinct signaling pathways differentially repress transcription and translation of proinflammatory cytokines such as TNFα and IL-6. Sustained endotoxin tolerance contributes to sepsis mortality. While transcription repression requires chromatin modifications, a translational repressor complex of Argonaute 2 (Ago2) and RNA-binding motif protein 4 (RBM4), which bind the 3'-UTR of TNFα and IL-6 mRNA, limits protein synthesis. Here, we show that Dcp1 supports the assembly of the Ago2 and RBM4 repressor complex into cytoplasmic processing bodies (p-bodies) in endotoxin-tolerant THP-1 human monocytes following stimulation with LPS, resulting in translational repression and limiting protein synthesis. Importantly, this translocation process is reversed by Dcp1 knockdown, which restores TNFα and IL-6 protein levels. We also find this translational repression mechanism in primary macrophages of septic mice. Because p-body formation is a critical step in mRNA translation repression, we conclude that Dcp1 is a major component of the translational repression machinery of endotoxin tolerance and may contribute to sepsis outcome., (© 2015 S. Karger AG, Basel.)

Mounting evidence points to roles for miRNA gene regulation in promoting development, function, and cell survival in the mammalian retina. However, little is known regarding which retinal genes are targets of miRNAs. Here, we employed a systematic, nonbiased, biochemical approach to identify targets of miRNA gene regulation in the bovine retina, a common model species for vision research. Using Argonaute high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation analysis, we identified 348 high-confidence miRNA target sites within 261 genes. This list was enriched in rod and cone photoreceptor genes and included 28 retinal disease genes, providing further evidence of a role of miRNAs in the pathology of blinding diseases.

Argonaute2 binds to a short guide RNA (microRNA or short interfering RNA) and guides RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. Here we identified and characterized Argonaute2 from black tiger shrimp Penaeus monodon (designated as PmAgo2). The full-length cDNA of PmAgo2 contained a 5' untranslated region (UTR) of 106 bp, an open reading frame (ORF) of 2616 bp and a 3' UTR of 123 bp. The predicted PmAgo2 protein is 99.4 KDa with the theoretical isoelectric point of 9.54. PmAgo2 shared the highest similarity of amino acid with Marsupenaeus japonicus Argonaute2 and Litopenaeus vannamei Argonaute2, at 69.0% and 68.5%, respectively. Phylogenic analysis showed PmAgo2 clustered with shrimp Argonaute2, and closed to the group of insects. Real-time quantitative PCR showed that PmAgo2 was widely expressed in almost all examined tissues except eyestalk, with high expression in lymph and haemocyte. mRNA expression also revealed that PmAgo2 was significantly up-regulated by Staphylococcus aureus and White Spot Syndrome Virus (WSSV) in hepatopancreas. Furthermore, our study also confirmed that dsRNA and ssRNA homologous poly (I:C) and R848 activated the expression of PmAgo2. The result indicated that PmAgo2 responded to both bacterial infection and viral infection, especially, it may induce an ssRNA-mediated RNAi with other core members of siRNA pathway in black tiger shrimp., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

PIWIL2, a member of PIWI/AGO family, is expressed in germline stem cells and precancerous stem cells, but not in adult somatic cells. PIWIL2 plays an important role in tumor development. It is considered as a cancer–testis antigen (CT80). It has been reported that the spliced fragment of PIWIL2, PL2L60, was widely expressed in cancer cell lines. In this study, HLA-A2-restricted epitopes from PL2L60 were predicted by online tools. To improve the activity of the native epitope, a candidate peptide P281 with potent binding affinity was chosen to investigate the modification strategy. A series of aromatic amino acids were introduced to substitute the first residue of P281. Then, we tested the binding affinity and stability of the peptide analogs and their ability to elicit specific immune responses both in vitro and in vivo. Our results indicated that the cytotoxic T lymphocytes (CTLs) induced by [4-Cl-Phe1]P281 could elicit more potent activities than that of P281 and other analogs. The CTLs induced by this analog could lyze target cells in HLA-A2-restricted and antigen-specific manners. [4-Cl-Phe1]P281 also showed the best resistance against degradation in human serum. In conclusion, the introduction of the unnatural amino acid, 4-Cl-Phe, into the first position could enhance the activity of the native epitope to induce cytotoxic T lymphocytes. It might be a good strategy to modify other promising native epitopes. The novel epitopes identified in this study could be used as novel candidates to the immunotherapy of HLA-A2 positive patients with tumors expressing PL2L60.

Donor T cells that respond to host alloantigens following allogeneic bone marrow transplantation (BMT) induce graft-versus-host (GVH) responses, but their molecular landscape is not well understood. MicroRNAs (miRNAs) regulate gene (mRNA) expression and fine-tune the molecular responses of T cells. We stimulated naive T cells with either allogeneic or nonspecific stimuli and used argonaute cross-linked immunoprecipitation (CLIP) with subsequent ChIP microarray analyses to profile miR responses and their direct mRNA targets. We identified a unique expression pattern of miRs and mRNAs following the allostimulation of T cells and a high correlation between the expression of the identified miRs and a reduction of their mRNA targets. miRs and mRNAs that were predicted to be differentially regulated in allogeneic T cells compared with nonspecifically stimulated T cells were validated in vitro. These analyses identified wings apart-like homolog (Wapal) and synaptojanin 1 (Synj1) as potential regulators of allogeneic T cell responses. The expression of these molecular targets in vivo was confirmed in MHC-mismatched experimental BMT. Targeted silencing of either Wapal or Synj1 prevented the development of GVH response, confirming a role for these regulators in allogeneic T cell responses. Thus, this genome-wide analysis of miRNA-mRNA interactions identifies previously unrecognized molecular regulators of T cell responses.

Like many other classical autoantibodies in systemic rheumatic diseases, anti-Su antibodies were originally defined by the double immunodiffusion assay in the early 80s. However, despite its high prevalence, only a few reports on anti-Su were published in the following years and the progress in characterizing the target antigens and clinical significance was slow, probably due to its inconsistent or poor reactivity in other standard immunoassays. In 2006 the target antigen was identified as the microRNA (miRNA)-binding protein Argonaute 2 (Ago2). Ago2 is a key component of the RNA-induced silencing complex enriched in cytoplasmic foci called GW bodies. Due to preferential reactivity of human autoantibodies with native antigens, immunoprecipitation is the only method to reliably detect anti-Su/Ago2 antibodies. Anti-Su/Ago2 does not appear to have disease specificity since it is found in 10-20% of patients with various rheumatic diseases, including systemic lupus erythematosus, scleroderma, polymyositis/dermatomyositis, and Sjögren's syndrome, as well as apparently healthy individuals at lower prevalence. The clinical significance and the mechanism of production of anti-Su/Ago2 remains to be clarified.

GW/P bodies contain two TNRC6A protein isoforms (GW182 and TNGW1) that function as translational repressors of mRNA through Ago2-mediated RNA silencing. Autoantibodies to GW/P body components GW182, Ge-1 and Ago2 have previously been correlated with clinical autoimmune diseases including neurological disease, Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis and primary biliary cirrhosis. No studies were published to date examining if patients with autoantibodies directed against GW/P bodies contain autoantibodies to the trinucleotide repeat (TNR) region of TNGW1, which differs from GW182 only by the addition of an N-terminal QP-rich 253 amino acid sequence. Our data show that 85.7% of GW/P body positive plasma contain autoantibodies to various epitopes in the TNR region of TNGW1. Given the association of neurological diseases with autoantibodies directed to the TNR region on exon 5 of TNRC6A, this study examined whether there were TNR expansions as described in other neurological diseases and/or mutations in the nucleotide sequence of the CAG/CCA/G-rich region in seven anti-GW/P body positive patients, six control and eight breast cancer patients. Although a TNR expansion was not identified, 28.6% of patients containing autoantibodies to the TNR of TNGW1 were shown to have a single nucleotide polymorphism (SNP) at c.344C > A in the CAG/CCA/G-rich region of TNRC6A, which when translated, would produce a protein variant of p.Pro115Gln. The amino acid change may alter the structure of TNGW1 and/or perturb its miRNA regulatory function although this has not been examined experimentally. A putative change in protein structure may lead to a loss of tolerance to the TNGW1 protein or result in a "neo-antigen" in patients containing the specific TNRC6A SNPs. Further studies of a larger cohort of GW/P body positive patients and structure-function relationships of the variant TNRC6A are required to fully understand the role that such SNPs play in GW/P body autoantibody production and/or pathogenesis of related autoimmune diseases.

Small hairpin RNAs (shRNAs) having duplex lengths of 25-29 bp are normally processed by Dicer into short interfering RNAs (siRNAs) before incorporation into the RNA-induced silencing complex (RISC). However, shRNAs of ≤ 19 bp [short shRNAs (sshRNAs)] are too short for Dicer to excise their loops, raising questions about their mechanism of action. sshRNAs are designated as L-type or R-type according to whether the loop is positioned 3' or 5' to the guide sequence, respectively. Using nucleotide modifications that inhibit RNA cleavage, we show that R- but not L-sshRNAs require loop cleavage for optimum activity. Passenger-arm slicing was found to be important for optimal functioning of L-sshRNAs but much less important for R-sshRNAs that have a cleavable loop. R-sshRNAs could be immunoprecipitated by antibodies to Argonaute-1 (Ago1); complexes with Ago1 contained both intact and loop-cleaved sshRNAs. In contrast, L-sshRNAs were immunoprecipitated with either Ago1 or Ago2 and were predominantly sliced in the passenger arm of the hairpin. However, 'pre-sliced' L-sshRNAs were inactive. We conclude that active L-sshRNAs depend on slicing of the passenger arm to facilitate opening of the duplex, whereas R-sshRNAs primarily act via loop cleavage to generate a 5'-phosphate at the 5'-end of the guide strand.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.

Background: The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored., Methodology/principal Findings: Two monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons., Conclusions/significance: In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.