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Recent reports attribute numerous regulatory functions to the nuclear paraspeckle-forming long noncoding RNA, nuclear enriched assembly transcript 1 (NEAT1), but the implications of its involvement in Parkinson's disease (PD) remain controversial. To address this issue, we assessed NEAT1 expression levels and cell type patterns in the substantia nigra (SN) from 53 donors with and without PD, as well as in interference tissue culture tests followed by multiple in-house and web-available models of PD. PCR quantification identified elevated levels of NEAT1 expression in the PD SN compared with control brains, an elevation that was reproducible across a multitude of disease models. In situ RNA hybridization supported neuron-specific formation of NEAT1-based paraspeckles at the SN and demonstrated coincreases of NEAT1 and paraspeckles in cultured cells under paraquat (PQ)-induced oxidative stress. Furthermore, neuroprotective agents, including fenofibrate and simvastatin, induced NEAT1 up-regulation, whereas RNA interference-mediated depletion of NEAT1 exacerbated death of PQ-exposed cells in a leucine-rich repeat kinase 2-mediated manner. Our findings highlight a novel protective role for NEAT1 in PD and suggest a previously unknown mechanism for the neuroprotective traits of widely used preventive therapeutics.-Simchovitz, A., Hanan, M., Niederhoffer, N., Madrer, N., Yayon, N., Bennett, E. R., Greenberg, D. S., Kadener, S., Soreq, H. NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress.

Plants deploy RNA silencing as a natural defence against invading viruses involving sequence-specific degradation of the viral RNAs. As a counter-defence strategy, viruses encode suppressor proteins that simultaneously target different steps of the silencing machinery. Tomato leaf curl Palampur virus (ToLCPalV) is a bipartite begomovirus in Geminiviridae family. It is responsible for significant reduction in the crop yield and quality. DNA-A of the virus encodes for six proteins whereas DNA-B codes for two proteins. In this study, all viral genes were screened for their role in suppression of green fluorescent protein (GFP) silencing in Nicotiana tabacum cv. Xanthi, employing agrobacterium based co-infiltration assay. The assay identified AC4 as a potential suppressor of RNA silencing. In addition, AC4 expression also suppressed virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in N. benthamiana. Potato virus X (PVX) mediated transient expression of the AC4 in N. benthamiana showed enhanced symptoms that include downward leaf curling, leaf puckering and tissue necrosis. Further, N. benthamiana lines stably expressing AC4 showed severe developmental abnormalities. Mutational analysis suggested that glycine at 2 nd position is essential for AC4 pathogenicity. Collectively, these findings demonstrate the role of ToLCPalV AC4 in viral pathogenesis, disease establishment and suppression of gene silencing., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders inherited in an autosomal dominant fashion. The SCAs result in progressive gait imbalance, incoordination of the limbs, speech changes, and oculomotor dysfunction, among other symptoms. Over the past few decades, significant strides have been made in understanding the pathogenic mechanisms underlying these diseases. Although multiple efforts using a combination of genetics and pharmacology with small molecules have been made towards developing new therapeutics, no FDA approved treatment currently exists. In this review, we focus on SCA1, a common SCA subtype, in which some of the greatest advances have been made in understanding disease biology, and consequently potential therapeutic targets. Understanding of the underlying basic biology and targets of therapy in SCA1 is likely to give insight into treatment strategies in other SCAs. The diversity of the biology in the SCAs, and insight from SCA1 suggests, however, that both shared treatment strategies and specific approaches tailored to treat distinct genetic causes of SCA are likely needed for this group of devastating neurological disorders.

In 2011, a hexanucleotide repeat expansion in the first intron of the C9orf72 gene was identified as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The proposed disease mechanisms include loss of C9orf72 function and gain of toxicity from the bidirectionally transcribed repeat-containing RNAs. Over the last few years, substantial progress has been made to determine the contribution of loss and gain of function in disease pathogenesis. The extensive body of molecular, cellular, animal, and human neuropathological studies is conflicted, but the predominance of evidence favors gain of toxicity as the main pathogenic mechanism for C9orf72 repeat expansions. Alterations in several downstream cellular functions, such as nucleocytoplasmic transport and autophagy, are implicated. Exciting progress has also been made in therapy development targeting this mutation, such as by antisense oligonucleotide therapies targeting sense transcripts and small molecules targeting nucleocytoplasmic transport, and these are now in phase 1 clinical trials.

Background: One concern with the adoption of RNAi-based genetically engineered (GE) crops is the potential harm to valued non-target organisms. Species of Coccinellidae (Coleoptera) are important natural enemies and might be exposed to the insecticidal dsRNA produced by the plant. To assess their susceptibility to dietary RNAi, we fed Adalia bipunctata and Coccinella septempunctata with a dsRNA designed to target the vATPase A of the western corn rootworm, Diabrotica virgifera virgifera (Dvv dsRNA). Specific dsRNAs designed to target the vATPase A of the two ladybird beetle species served as positive controls., Results: Our results revealed that both species were sensitive to dietary RNAi when ingesting their own dsRNAs, with C. septempunctata being more sensitive than A. bipunctata. Dvv dsRNA also adversely affected the two ladybird beetles as indicated by a significantly (but marginally) prolonged developmental time for A. bipunctata and a significantly reduced survival rate for C. septempunctata. These results, however, were obtained at Dvv dsRNA concentrations that were orders of magnitude higher than expected to occur in the field. Gene expression analyses confirmed the bioactivity of the dsRNA treatments and the results from the feeding bioassays. These results are consistent with the bioinformatics analyses, which revealed a higher number of 21-nucleotide-long matches, a requirement for effective RNAi, of the Dvv dsRNA with the vATPase A of C. septempunctata (34 matches) than with that of A. bipunctata (six matches)., Conclusion: Feeding bioassays revealed that two ladybird species are responsive to dietary RNAi. The two species, however, differed in their sensitivity. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

RNA interference (RNAi) has strong antiviral activity in a range of animal phyla, but the extent to which RNAi controls virus infection in chordates, and specifically mammals remains incompletely understood. Here we analyze the antiviral activity of RNAi against a number of positive-sense RNA viruses using Argonaute-2 deficient human cells. In line with absence of virus-derived siRNAs, Sindbis virus, yellow fever virus, and encephalomyocarditis virus replicated with similar kinetics in wildtype cells and Argonaute-2 deficient cells. Coxsackievirus B3 (CVB3) carrying mutations in the viral 3A protein, previously proposed to be a virus-encoded suppressor of RNAi in another picornavirus, human enterovirus 71, had a strong replication defect in wildtype cells. However, this defect was not rescued in Argonaute-2 deficient cells, arguing against a role of CVB3 3A as an RNAi suppressor. In agreement, neither infection with wildtype nor 3A mutant CVB3 resulted in small RNA production with the hallmarks of canonical vsiRNAs. Together, our results argue against strong antiviral activity of RNAi under these experimental conditions, but do not exclude that antiviral RNAi may be functional under other cellular, experimental, or physiological conditions in mammals.

The stability and quality of metazoan mRNAs are under microRNA (miRNA)-mediated and nonsense-mediated control. Although UPF1, a core mediator of nonsense-mediated mRNA decay (NMD), mediates the decay of target mRNA in a 3'UTR-length-dependent manner, the detailed mechanism remains unclear. Here, we suggest that 3'UTR-length-dependent mRNA decay is not mediated by nonsense mRNAs but rather by miRNAs that downregulate target mRNAs via Ago-associated UPF1/SMG7. Global analyses of mRNAs in response to UPF1 RNA interference in miRNA-deficient cells reveal that 3'UTR-length-dependent mRNA decay by UPF1 requires canonical miRNA targeting. The destabilization of miRNA targets is accomplished by the combination of Ago2 and UPF1/SMG7, which may recruit the CCR4-NOT deadenylase complex. Indeed, loss of the SMG7-deadenylase complex interaction increases the levels of transcripts regulated by UPF1-SMG7. This UPF1/SMG7-dependent miRNA-mediated mRNA decay pathway may enable miRNA targeting to become more predictable and expand the miRNA-mRNA regulatory network.

Plants are exposed every day to multiple environmental cues, and tight transcriptome reprogramming is necessary to control the balance between responses to stress and processes of plant growth. In this context, the silencing phenomena mediated by small RNAs can drive transcriptional and epigenetic regulatory modifications, in turn shaping plant development and adaptation to the surrounding environment. Mounting experimental evidence has recently pointed to small noncoding RNAs as fundamental players in molecular signalling cascades activated upon exposure to abiotic and biotic stresses. Although, in the last decade, studies on stress responsive small RNAs increased significantly in many plant species, the physiological responses triggered by these molecules in the presence of environmental stresses need to be further explored. It is noteworthy that small RNAs can move either cell-to-cell or systemically, thus acting as mobile silencing effectors within the plant. This aspect has great importance when physiological changes, as well as epigenetic regulatory marks, are inspected in light of plant environmental adaptation. In this review, we provide an overview of the categories of mobile small RNAs in plants, particularly focusing on the biological implications of non-cell autonomous RNA silencing in the stress adaptive response and epigenetic modifications.

Uridine insertion deletion editing in kinetoplastid protozoa requires a complex machinery, a primary component of which is the RNA editing substrate binding complex (RESC). RESC contains two modules termed GRBC (guide RNA binding complex) and REMC (RNA editing mediator complex), although how interactions between these modules and their mRNA and gRNA binding partners are controlled is not well understood. Here, we demonstrate that the ARM/HEAT repeat containing RESC protein, MRB10130, controls REMC association with mRNA- and gRNA-loaded GRBC. High-throughput sequencing analyses show that MRB10130 functions in both initiation and 3' to 5' progression of editing through gRNA-defined domains. Editing intermediates that accumulate upon MRB10130 depletion significantly intersect those in cells depleted of another RESC organizer, MRB7260, but are distinct from those in cells depleted of specific REMC proteins. We present a model in which MRB10130 coordinates numerous protein-protein and protein-RNA interactions during editing progression., (© 2019 McAdams et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

In a reductionist perspective, plant silencing small (s)RNAs are often classified as mediating nuclear transcriptional gene silencing (TGS) or cytosolic posttranscriptional gene silencing (PTGS). Among the PTGS diagnostics is the association of AGOs and their sRNA cargos with the translation apparatus. In Arabidopsis , this is observed for AGO1 loaded with micro(mi)RNAs and, accordingly, translational-repression (TR) is one layer of plant miRNA action. Using AGO1:miRNA-mediated TR as a paradigm, we explored, with two unrelated polysome-isolation methods, which, among the ten Arabidopsis AGOs and numerous sRNA classes, interact with translation. We found that representatives of all three AGO-clades associate with polysomes, including the TGS-effector AGO4 and stereotypical 24-nt sRNAs that normally mediate TGS of transposons/repeats. Strikingly, approximately half of these annotated 24-nt siRNAs displayed unique matches in coding regions/introns of genes, and in pseudogenes, but not in transposons/repeats commonly found in their vicinity. Protein-coding gene-derived 24-nt sRNAs correlate with gene-body methylation. Those derived from pseudogenes belong to two main clusters defined by their parental-gene expression patterns, and are vastly enriched in AGO5, itself found on polysomes. Based on their tight expression pattern in developing and mature siliques, their biogenesis, and genomic/epigenomic features of their loci-of-origin, we discuss potential roles for these hitherto unknown polysome-enriched, pseudogene-derived siRNAs., (© 2019 Marchais et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Silent corticotroph tumors are a pituitary neuroendocrine tumor subtype of corticotroph lineage that do not clinically express Cushing's disease. The silencing of this type of tumor is not fully understood. The aim of the present study was to delve into the lack of secretory activity, studying the post-transcriptional and post-translational regulation of POMC/ACTH in a series of molecularly identified functioning and silent corticotroph tumors., Design: We analyzed 24 silent corticotroph, 23 functioning corticotroph and 25 silent gonadotroph tumors., Methods: We used Sanger sequencing, quantitative real-time PCR and Western blot to analyze genetic alterations in POMC, gene expression of TBX19, NEUROD1, POMC, PCSK1, PCSK2, CPE and PAM and protein expression of POMC, PC1/3, PC2, CPE and PAM., Results: We found different polymorphisms in the POMC gene of corticotroph tumors, some of them related to deficiency of proopiomelanocortin. Silent corticotroph tumors showed lower PC1/3 gene and protein expression than functioning ones, especially compared to micro-functioning corticotroph tumors (all P < 0.05). Moreover, we found a positive correlation between PC2 and CPE gene and protein expression (rho ≥ 0.670, P < 0.009) in silent corticotroph tumors compared with functioning ones., Conclusions: By studying the post-transcriptional and post-translational processing of POMC and ACTH, respectively, in a large series of silent and functioning corticotroph tumors, we found that the lack of secretory activity of these tumors is related to an impaired processing of POMC and a high degradation of ACTH, with the macro-functioning corticotroph tumor behaving as an intermediate state between micro-functioning and silent corticotroph tumors.

Long non-coding RNAs (lncRNAs), are over 200 nucleotides in length, and they rarely act as templates for protein synthesis. Mounting studies have shown that lncRNAs play a crucial regulatory role in various processes that sustain life, such as epigenetic regulation, cell cycle control, splicing, and post-transcriptional regulation. LncRNAs were aberrantly expressed in most hematological malignancies including lymphoma, participating in tumor suppression or promoting oncogenesis and modulating key genes in different pathways. The specific expression patterns of lncRNAs in lymphoma make them good candidates to be used as diagnostic biomarkers or as therapeutic targets. LncRNAs can be targeted by multiple approaches including nucleic acid therapeutics, CRISPR/Cas genome editing techniques, small molecule inhibitors, and gene therapy. Efforts are made to develop therapeutic strategies aimed at targeting lncRNAs, but there are still some avenues to be covered before they can be applied to the clinical treatment of lymphoma., (Copyright © 2019 Elsevier B.V. All rights reserved.)

RNA interference (RNAi) is a potentially useful pest control method because of its high specificity. Silencing the expression of important RNAi target genes of pests will block important biological processes and reduce pest damage. Ecdysone is a unique arthropod hormone and the ecdysone receptor (EcR) is a key factor in molting pathway. We investigated the possibility that dsRNA targeting of the EcR of Tetranychus cinnabarinus (TcEcR) could effectively block development from larvae to adults. The mRNA level of TcEcR was highest in the larva stage, and 73.1% of the mites failed to survive the larva stage when TcEcR expression was silenced. Only 11.7% of T. cinnabarinus ingesting dsRNA successfully developed into adults, while 86.7% in the control succeeded in molting across each stage. RNAi significantly increased the developmental intervals of T. cinnabarinus. Under the effects of dsRNA, development times for the larva and first nymph doubled. Phenotype of body size change and death were observed during the development of T. cinnabarinus ingesting dsRNA. These findings suggest that RNAi is a potential means for the control of T. cinnabarinus. Genes in hormone pathways such as EcR are possible RNAi targets., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Small interfering RNA (siRNA) has found many applications in tissue regeneration and disease therapeutics. Effective and localized siRNA delivery remains challenging, reducing its therapeutic potential. Here, we report a strategy to control and prolong siRNA release by directly tethering transfection-capable siRNA to photocrosslinked dextran hydrogels. siRNA release is governed via the hydrolytic degradation of ester and/or disulfide linkages between the siRNA and hydrogels, which is independent of hydrogel degradation rate. The released siRNA is shown to be bioactive by inhibiting protein expression in green fluorescent protein-expressing HeLa cells without the need of a transfection agent. This strategy provides an excellent platform for controlling nucleic acid delivery through covalent bonds with a biomaterial and regulating cellular gene expression, which has promising potential in many biomedical applications.

The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.

Small interfering RNA (siRNA) is considered a highly specific and potent biotherapeutic that holds tremendous potential for the treatment of various diseases. The clinical translation of siRNA is, however, greatly impeded by the lack of safe and efficient delivery vehicles in vivo. Here, the development of selective cell penetrating peptide (CPP33)-functionalized chimeric lipopepsomes (CPP33-CLP) for efficient encapsulation and selective delivery of polo-like kinase 1 specific siRNA (siPLK1) to orthotopic A549 human lung tumor in vivo is reported. Interestingly, siRNA is tightly encapsulated into CPP33-CLP with a superb encapsulation efficiency of over 95% owing to the thick strong electrostatic interactions. Notably, siPLK1-loaded CPP33-CLP (siPLK1-CPP33-CLP) is selectively internalized by A549 human lung cancer cells, efficiently escapes from endosomes, and swiftly releases siRNA into the cytoplasm, affording a significant sequence-specific gene silencing in vitro. Moreover, siPLK1-CPP33-CLP exhibits prolonged blood circulation, enhanced tumor accumulation, effective suppression of tumor growth, and considerably elevated survival time of orthotopic A549 human lung tumor-bearing nude mice. These chimeric lipopepsomes appear as an attractive and potent nanoplatform for safe and targeted siRNA delivery., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The discovery of small RNAs has offered exciting opportunities in manipulating gene expression. The non-coding RNAs cause target gene inactivation at the transcriptional, post-transcriptional or translational level. In addition to the default silencing approach, they provide another mode of gene regulation by transitivity. Here, gradual amplification in effector RNAs number allows regulation of genes other than the original target and causes the outspread of silencing from its origin to aid a robust response. Unlike the short-range cell-to-cell movement of silencing signal (through plasmodesmata), little is known of the mediators of systemic silencing (usually through phloem). Through the present review, we combine the reports available so far to better understand the characteristics of secondary silencing, factors involved, and summarize the instances where it has been employed in plants. Understanding the molecular mechanism behind transitivity has led to the designing of efficient transgenes for targeted gene inactivation, utilized in silencing of a multigene family, and in the field of functional genomics. Studies uncovering the origin of distinct secondary silencing pathways in plants have been exploited for developing artificial RNA silencing methods such as hairpin RNA, artificial microRNA, intrinsic direct repeat, inverted repeat, artificial trans-acting siRNA, phased siRNA, and virus-induced gene silencing. The techniques have facilitated the spread of traits such as pathogenic resistance or alter plant growth and development features. The mechanism of reprogramming in the silencing machinery and the consequent genetic manipulation through transitive RNA is still not completely understood and its exploitation in crop improvement programmes is still in a developing phase.

Long noncoding RNA maternally expressed gene 3 (lncRNA-MEG3) is an important regulator in multiple biological functions. However, lncRNA-MEG3's function in cattle growth and regulatory mechanism on bovine skeletal muscle development has not yet been well studied. In this project, we first investigated lncRNA-MEG3's expression profile and detected that it was highly expressed in bovine skeletal muscle tissue and its RNA level was kept increasingly during the early phase of bovine primary myoblast differentiation. Using luciferase reporter assays, we identified the lncRNA-MEG3 core promoter containing putative transcription factor binding site for myocyte enhancer factor 2C (MEF2C). Interestingly, we found that LncRNA-MEG3 could significantly upregulate and downregulate myosin heavy chain ( MHC), myogenin ( MyoG), and MEF2C through overexpression and RNAi strategies, respectively. Using luciferase reporter assays, we also verified lncRNA-MEG3 as a miR-135 sponge. Overexpression of miR-135 markedly inhibited the wild type of lncRNA-MEG3, but not the mutant lncRNA-MEG3 reporter. The luciferase activity of miR-135 sensor could be rescued by lncRNA-MEG3 via competing for miRNA-135. In addition, the luciferase activity of MEF2C was significantly upregulated by the wild type of lncRNA-MEG3. This study, for the first time, revealed that lncRNA-MEG3 could promote bovine skeletal muscle differentiation via interacting with miRNA-135 and MEF2C. The results were valuable for further studies and applications of lncRNA related roles in beef molecular breeding., (© 2019 Wiley Periodicals, Inc.)

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3'UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas, and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identified as the predominant miRNA with effects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.

Objectives: Long non-coding RNAs (LncRNAs) play an important role in hepatocellular carcinoma development, however, as a crucial driver of hepatocellular carcinoma (HCC) metastasis, their functions in tumour metastasis remain largely unknown., Materials and Methods: The lncRNA TRERNA1 expression levels were detected in HCC by quantitative real-time PCR (qPCR). The function of TRERNA1 was examined by wound-healing assays, transwell assays and tail vein injection experiments. The potential regulatory mechanisms of TRERNA1 on its target genes were explored by ChIP, RIP, IP and WB assays., Results: Elevated TRERNA1 levels promoted HCC cell migration and invasion in vitro and in vivo. TRERNA1 recruited EHMT2 to dimethylate H3K9 in the CDH1 promoter region. Furthermore, EHMT2 bound to SNAI1 to suppress CDH1 expression in HCC cells. After inhibiting TRERNA1, the expression level of CDH1 was restored and was involved in the regulation of the EHMT2/SNAI1 complex. The level of TRERNA1 was positively correlated with tumour metastasis and was negatively correlated with the expression of CDH1 in HCC tissues., Conclusions: For the first time, the current study reveals that TRERNA1 promotes cell metastasis and the invasion of HCC via the recruitment of EHMT2 and/or the EHMT2/SNAI1 complex to suppress CDH1. These data identify a novel mechanism that regulates TRERNA1 in metastatic HCC and provides a potential targeted therapy for HCC patients., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Long-noncoding RNAs (lncRNAs) is involved in the development of diverse diseases, including leukemia, while the role lncRNA HOX transcript antisense RNA (HOTAIR) played in leukemia remains unclear and in need of further investigation. Therefore, this study was conducted to explore the effects of lncRNA HOTAIR on the immunologic rejection of leukemia cells through the Wnt/β-catenin in mice. Mice were administrated with HOTAIR mimics as well as small interfering RNA HOTAIR to explore the regulatory role of HOTAIR. The numbers of white blood cell (WBC) and platelet (PLT) and the content of hemoglobin in peripheral blood (PB) were determined. The cytokine level in PB was measured. T-lymphocyte proliferation activity, Ig production by B cells, natural killer (NK) cell activity, and the proportion of cluster of differentiation 4 (CD4)/CD8 T cell subsets were detected. Expression of HOTAIR, β-catenin, cyclinD1, GSK-3β, and c-Myc in bone marrow was determined. It was revealed that the WBC number increased, while the PLT number along with the hemoglobin content in PB decreased with the upregulated HOTAIR. Additionally, elevated HOTAIR led to decreased levels of transforming growth factor-β, interferon-γ, interleukin-10, and tumor necrosis factor-α in PB, proliferation activity in T-lymphocyte, and inhibited Ig production, NK cell activity, and the ratio of CD4/CD8 T cell subsets in B-lymphocyte. Furthermore, Wnt/β-catenin was activated by overexpressing HOTAIR. Enhanced survival and proliferation were shown with increased expression of cyclinD1, GSK-3β, and c-Myc in the bone marrow of mice induced by overexpressing HOTAIR. These results indicate that restored HOTAIR reduces the immunologic rejection of leukemia cells in mice by activating Wnt/β-catenin pathway., (© 2019 Wiley Periodicals, Inc.)

As detoxification enzymes, proteins in the glutathione S-transferase (GST) superfamily are reported to participate in oxidative stress resistance. Nevertheless, microsomal GSTs (MGSTs), a unique subclass of the GST superfamily associated with membranes, are rarely studied in insects. Here, we isolated an MGST gene in Apis cerana cerana (AccMGST1) and verified its role in oxidative stress response. We found higher expression of AccMGST1 in protective or defensive tissue, that is, the epidermis, which indicated its role in stress resistance. Real-time quantitative PCR (qRT-PCR) analysis indicated that AccMGST1 was upregulated by oxidative stresses at the transcriptional level. In contrast, AccMGST1 expression was inhibited when the antioxidant vitamin C (VC) was fed to experimental bees. Through western blotting, we found that the protein level of AccMGST1 under oxidative stress corresponded to the transcript level. Disc diffusion and mixed-function oxidation (MFO) assays suggested that AccMGST1 can protect not only cells but also DNA against oxidative damage. Furthermore, we discovered that the expression patterns of known antioxidant genes were changed in A. cerana cerana after AccMGST1 was silenced by RNA interference (RNAi). Thus, we concluded that the gene AccMGST1 exerts a significant role in the antioxidant mechanism.

Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents re-replication in vertebrates and invertebrates. Previously, we identified two Geminin genes, BmGeminin1 and BmGeminn2 , in the silkworm Bombyx mori , and we found that RNA interference of BmGeminin1 led to re-replication. However, the function of BmGeminin2 remains poorly understood. In this study, we found that knockdown of BmGeminin2 can improve cell proliferation, and upregulated G2/M-associated gene- cyclinB / CDK1 expression. Then, we performed yeast two-hybrid screening to identify interacting proteins. Our results yielded 23 interacting proteins, which are involved in DNA replication, chromosome stabilization, embryonic development, energy, defense, protein processing, or structural protein. Here, we focused on BmRRS1, a chromosome congression-related protein that is closely related to cell cycle G2/M progression. The interaction between BmGeminin2 and BmRRS1 was confirmed by immunofluorescence and immunoprecipitation. Analysis of its expression profile showed that BmRRS1 was related to BmGeminin2 . In addition, BmGeminin2 overexpression downregulated the BmRRS1 transcript. Knockdown of BmGeminin2 led to upregulation of the BmRRS1 transcript. Furthermore, overexpression of BmRRS1 can upregulate G2/M-associated gene- cyclinB / CDK1 expression, and improved cell proliferation, consistent with the effects of BmGeminin2 knockout. In addition, BmRRS1 RNA interference can eliminate the impact of BmGem2 knockout on cell proliferation, the ratio of cell cycle stage and the expression of cyclinB / CDK1 . These data suggested that the cell proliferation advantage of BmGeminin2 knockout was closely related to BmRRS1 . Our findings provide insight into the functions of Geminin and the mechanisms underlying the regulation of the cell cycle in the silkworm.

Tumour-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumour cells that are now understood to facilitate communication between the tumour and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-GTP following nuclear export and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-GTP and GRP1. As such, ARF6 activation increases the pre-miRNA cargo contained within TMVs through a process that requires the casein kinase 2-mediated phosphorylation of RanGAP1. Furthermore, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute-2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.

RNAi experiments are ubiquitously used in cell biology and are achieved by transfection of small interfering RNAs (siRNAs) into cells using a transfection reagent. These results in knock-down of proteins of interest, and the phenotypic consequences are then analyzed. It is reported here that two common RNA interference (RNAi) transfection reagents, DharmaFECT 1 and INTERFERin, in mock transfections using non-targeting siRNAs, cause alterations in the lipidome of HeLa cells. Some lipids change in response to both, presumably chemically different, transfection reagents, while other lipid species change only in response to one of the reagents. While the functional implications of these lipidomic alterations remain to be investigated, the authors' experiments suggest that it is important to use appropriate mock transfection controls during RNAi experiments, ideally complemented by an orthogonal perturbation, especially when investigating membrane-associated phenomena., (© 2019 The Authors. Proteomics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

During suboptimal growth conditions, Caenorhabditis elegans larvae undergo a global developmental arrest called "dauer." During this stage, the germline stem cells (GSCs) become quiescent in an AMP-activated Protein Kinase (AMPK)-dependent manner, and in the absence of AMPK, the GSCs overproliferate and lose their reproductive capacity, leading to sterility when mutant animals resume normal growth. These defects correlate with the altered abundance and distribution of a number of chromatin modifications, all of which can be corrected by disabling components of the endogenous small RNA pathway, suggesting that AMPK regulates germ cell integrity by targeting an RNA interference (RNAi)-like pathway during dauer. The expression of AMPK in somatic cells restores all the germline defects, potentially through the transmission of small RNAs. Our findings place AMPK at a pivotal position linking energy stress detected in the soma to a consequent endogenous small RNA-mediated adaptation in germline gene expression, thereby challenging the "permeability" of the Weismann barrier., Competing Interests: The authors have declared that no competing interests exist.

Grain starch and protein are synthesized during endosperm development, prompting the question of what regulatory mechanism underlies the synchronization of the accumulation of secondary and primary gene products. We found that two endosperm-specific NAC transcription factors, ZmNAC128 and ZmNAC130, have such a regulatory function. Knockdown of expression of ZmNAC128 and ZmNAC130 with RNA interference (RNAi) caused a shrunken kernel phenotype with significant reduction of starch and protein. We could show that ZmNAC128 and ZmNAC130 regulate the transcription of Bt2 and then reduce its protein level, a rate-limiting step in starch synthesis of maize endosperm. Lack of ZmNAC128 and ZmNAC130 also reduced accumulation of zeins and nonzeins by 18% and 24% compared with nontransgenic siblings, respectively. Although ZmNAC128 and ZmNAC130 affected expression of zein genes in general, they specifically activated transcription of the 16-kDa γ-zein gene. The two transcription factors did not dimerize with each other but exemplified redundancy, whereas individual discovery of their function was not amenable to conventional genetics but illustrated the power of RNAi. Given that both the Bt2 and the 16-kDa γ-zein genes were activated by ZmNAC128 or ZmNAC130, we could identify a core binding site ACGCAA contained within their target promoter regions by combining Dual-Luciferase Reporter and Electrophoretic Mobility Shift assays. Consistent with these properties, transcriptomic profiling uncovered that lack of ZmNAC128 and ZmNAC130 had a pleiotropic effect on the utilization of carbohydrates and amino acids., Competing Interests: The authors declare no conflict of interest.

Autophagy is a conserved degradation process that occurs in all eukaryotic cells and its dysfunction has been associated with various diseases including cancer. While a number of large-scale attempts have recently identified new molecular players in autophagy regulation, including proteins and microRNAs, little is known regarding the function of long non-coding RNAs (lncRNAs) in the regulation of this process. To identify new long non-coding RNAs with functional implications in autophagy, we performed a high-throughput RNAi screen targeting more than 600 lncRNA transcripts and monitored their effects on autophagy in MCF-7 cells. We identified 63 lncRNAs that affected GFP-LC3B puncta numbers significantly. We validated the strongest hit, the lncRNA DRAIC previously shown to impact cell proliferation, and revealed a novel role for this lncRNA in the regulation of autophagic flux. Interestingly, we find DRAIC's pro-proliferative effects to be autophagy-independent. This study serves as a valuable resource for researchers from both the lncRNA and autophagy fields as it advances the current understanding of autophagy regulation by non-coding RNAs.

Gene silencing by RNA interference is extensively used reverse genetic approach to analyse the implications of any gene in mammalian systems. The silencing of the Activin type IIB receptor belonging to transforming growth factor beta superfamily has demonstrated increase in muscle growth in many species. We designed five short hairpin RNA constructs targeting coding region of chicken ACTRIIB. All the shRNAs were transfected into chicken embryo fibroblast cells and evaluated their silencing efficiency by real time PCR and western blotting. Initially the computational analysis of target region and shRNA constructs was undertaken to predict sequence based features (secondary structures, GC% and H-b index) and thermodynamic features (ΔG overall , ΔG duplex , ΔG break-target , ΔG intra-oligomer , ΔG inter-oligomer and ΔΔG ends ). We determined that all these predicted features were associated with shRNA efficacy. The invitro analysis of shRNA constructs exhibited significant (P < 0.05) reduction in the levels of ACTRIIB at mRNA and protein level. The knock down efficiency of shRNAs varied significantly (P < 0.001) from 83% (shRNA 1) to 43% (shRNA 5). All the shRNAs up regulated the myogenic pathway associated genes (MyoD and MyoG) significantly (P < 0.05). There was significant (P < 0.05) up-regulation of IFNA, IFNB and MHCII transcripts. The ACTRIIB expression was inversely associated with the expression of myogenic pathway and immune response genes. The anti ACTRIIB shRNA construct 1 and 3 exhibited maximum knock down efficiency with minimal interferon response, and can be used for generating ACTRIIB knockdown chicken with higher muscle mass.

Angiogenesis is critical to cancer development and metastasis. However, anti-angiogenic agents have only had modest therapeutic success, partly due to an incomplete understanding of tumor endothelial cell (EC) biology. We previously reported that the microRNA (miR)-200 family inhibits metastasis through regulation of tumor angiogenesis, but the underlying molecular mechanisms are poorly characterized. Here, using integrated bioinformatics approaches, we identified the RNA-binding protein (RBP) quaking (QKI) as a leading miR-200b endothelial target with previously unappreciated roles in the tumor microenvironment in lung cancer. In lung cancer samples, both miR-200b suppression and QKI overexpression corresponded with tumor ECs relative to normal ECs, and QKI silencing phenocopied miR-200b-mediated inhibition of sprouting. Additionally, both cancer cell and endothelial QKI expression in patient samples significantly corresponded with poor survival and correlated with angiogenic indices. QKI supported EC function by stabilizing cyclin D1 (CCND1) mRNA to promote EC G1/S cell cycle transition and proliferation. Both nanoparticle-mediated RNA interference of endothelial QKI expression and palbociclib blockade of CCND1 function potently inhibited metastasis in concert with significant effects on tumor vasculature. Altogether, this work demonstrates the clinical relevance and therapeutic potential of a novel, actionable miR/RBP axis in tumor angiogenesis and metastasis.

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60-80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T 4 ) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T 4 seeds compared to NT seed controls. A significant increase in the contents of Fe 2+ (15.4%, 21.7%), Zn 2+ (7.45%, 11.15%) and Ca 2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

The RNA interference (RNAi) pathway is a potent antiviral defense mechanism in plants and invertebrates, in response to which viruses evolved suppressors of RNAi. In mammals, the first line of defense is mediated by the type I interferon system (IFN); however, the degree to which RNAi contributes to antiviral defense is still not completely understood. Recent work suggests that antiviral RNAi is active in undifferentiated stem cells and that antiviral RNAi can be uncovered in differentiated cells in which the IFN system is inactive or in infections with viruses lacking putative viral suppressors of RNAi. In this review, we describe the mechanism of RNAi and its antiviral functions in insects and mammals. We draw parallels and highlight differences between (antiviral) RNAi in these classes of animals and discuss open questions for future research.

Systemic RNAi, initiated by double-stranded RNA (dsRNA) ingestion, has been reported in diverse invertebrates, including honey bees, demonstrating environmental RNA uptake that undermines homologous gene expression. However, the question why any organism would take up RNA from the environment has remained largely unanswered. Here, we report on horizontal RNA flow among honey bees mediated by secretion and ingestion of worker and royal jelly diets. We demonstrate that transmission of jelly-secreted dsRNA to larvae is biologically active and triggers gene knockdown that lasts into adulthood. Worker and royal jellies harbor differential naturally occurring RNA populations. Jelly RNAs corresponded to honey bee protein-coding genes, transposable elements, and non-coding RNA, as well as bacteria, fungi, and viruses. These results reveal an inherent property of honey bees to share RNA among individuals and generations. Our findings suggest a transmissible RNA pathway, playing a role in social immunity and signaling between members of the hive., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Novel therapeutic strategies for non-small-cell lung cancer (NSCLC) are urgently needed. RNA splicing, orchestrated by the spliceosome, is deregulated in many forms of cancer, including NSCLC. Here, we performed high-throughput screening with a small interfering RNA library targeting all annotated human spliceosome proteins to identify cancer-selective lethal targets in the RNA splicing machinery. Silencing of several spliceosome proteins reduced cell viability in a panel of NSCLC cell lines, but not in non-malignant lung fibroblasts and epithelial cells. Interestingly, the cancer-selective lethal target set comprised all seven Sm proteins that, together with small nuclear RNA, form the core structure of most spliceosome subunits. Interfering with Sm protein expression induced apoptosis in NSCLC cells, but not in non-malignant cells. In silico analysis revealed that Sm proteins are frequently upregulated in NSCLC. For several Sm proteins, increased expression showed a positive correlation with disease severity. Together, our results suggest that the Sm proteins represent particularly useful novel targets for selective treatment of NSCLC.

Delivery of RNAi-mediating shRNA molecules for gene silencing via bacteria, i.e. by transkingdom RNAi (tkRNAi) technology, is suggested to be a powerful alternative technique. In this work, the efficiency of bacterial delivery of shRNAs directed against HPV16-E7-specific mRNA to oral squamous carcinoma cells (OSCCs) was evaluated. E. coli were transfected with a plasmid encoding the inv locus and the Hlya gene to enable the bacteria to enter carcinoma cells and to escape from endocytotic vesicles. The bacterial penetration to the target cells was confirmed by DAPI staining. The HPV16-E7 mRNA expression in bacteria-treated OSCCs dropped to 61% of the controls as measured by qRT-PCR. Corresponding inhibition of the HPV16-E7 protein was confirmed by western blotting. The IC 50 of bacteria-treated OSCCs was reduced to more than 75%. Flow cytometry assays showed higher total apoptosis and caspase-3 activation (6.6-fold and 8.4-fold respectively) in OSCCs following exposure to anti-HPV-E7  bacteria compared to anti-GFP bacteria (2-fold and 2.9-fold, respectively). In conclusion, it was demonstrated for the first time that tkRNAi technology is also useful for treatment of squamous carcinoma cells. Anti-HPV16-E7 shRNA-encoding bacteria can efficiently deliver RNAi effectors to OSCCs mediating a strong and specific gene silencing associated with triggering cell death.

Background: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries., Objective: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library., Methods: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis., Results: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences., Conclusion: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gα z protein - a signalling molecule within the G-protein coupled receptor network. Our findings suggest that GNAZ/Gα z contribute to the MCL pathobiology., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion.

The gene daf-12 has long shown to be involved in the dauer pathway in Caenorhabditis elegans (C. elegans). Due to the similarities of the dauer larvae of C. elegans and infective larvae of certain parasitic nematodes such as Strongyloides spp., this gene has also been suspected to be involved in the development of infective larvae. Previous research has shown that the application of dafachronic acid, the steroid hormone ligand of DAF-12 in C. elegans, affects the development of infective larvae and metabolism in Strongyloides. However, a lack of tools for either forward or reverse genetics within Strongyloides has limited studies of gene function within these important parasites. After determining whether Strongyloides had the requisite proteins for RNAi, we developed and report here the first successful RNAi by soaking protocol for Strongyloides ratti (S. ratti) and use this protocol to study the functions of daf-12 within S. ratti. Suppression of daf-12 in S. ratti severely impairs the formation of infective larvae of the direct cycle and redirects development towards the non-infective (non-dauer) free-living life cycle. Further, daf-12(RNAi) S. ratti produce slightly but significantly fewer offspring and these offspring are developmentally delayed or incapable of completing their development to infective larvae (L3i). Whilst the successful daf-12(RNAi) L3i are still able to infect a new host, the resulting infection is less productive and shorter lived. Further, daf-12 knockdown affects metabolism in S. ratti resulting in a shift from aerobic towards anaerobic fat metabolism. Finally, daf-12(RNAi) S. ratti have reduced tolerance of temperature stress., Competing Interests: The authors have declared that no competing interests exist.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase., Competing Interests: The authors have declared that no competing interests exist.

In budding yeast, which possesses simple point centromeres, we discovered that all of its centromeres express long noncoding RNAs (cenRNAs), especially in S phase. Induction of cenRNAs coincides with CENP-A Cse4 loading time and is dependent on DNA replication. Centromeric transcription is repressed by centromere-binding factor Cbf1 and histone H2A variant H2A.Z Htz1 Deletion of CBF1 and H2A.Z HTZ1 results in an up-regulation of cenRNAs; an increased loss of a minichromosome; elevated aneuploidy; a down-regulation of the protein levels of centromeric proteins CENP-A Cse4 , CENP-A chaperone HJURP Scm3 , CENP-C Mif2 , Survivin Bir1 , and INCENP Sli15 ; and a reduced chromatin localization of CENP-A Cse4 , CENP-C Mif2 , and Aurora B Ipl1 When the RNA interference system was introduced to knock down all cenRNAs from the endogenous chromosomes, but not the cenRNA from the circular minichromosome, an increase in minichromosome loss was still observed, suggesting that cenRNA functions in trans to regulate centromere activity. CenRNA knockdown partially alleviates minichromosome loss in cbf1Δ , htz1Δ , and cbf1Δ htz1Δ in a dose-dependent manner, demonstrating that cenRNA level is tightly regulated to epigenetically control point centromere function., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

'HoneySweet', a transgenic plum (Prunus domestica) resistant to plum pox virus through RNAi, was deregulated in the U.S. in 2011. The compositional study of 'HoneySweet' fruit was expanded to include locations outside of the US as well as utilizing a wide variety of comparators and different collection years to see the variability possible. The results revealed that plums have a wide variation in composition and that variation among locations was greater than variation among cultivars. This was also the case for different years at one location. The results supported the supposition that the transgene and insertion event had no significant effect on the composition of 'HoneySweet' fruit even under virus pressure, and that it fell in the normal range of composition of commercially grown plums. It also suggested that the effect of environment is as great as that of genetics on the fruit composition of plums., Competing Interests: No competing interests are declared. ‘HoneySweet’ plum is patented (USPP15154P2, 2004) jointly by Institut National de la Recherche Agronomique (INRA), Cornell Research Foundation Inc and the US Department of Agriculture with Ralph Scorza listed as one of the inventors. It is freely available. Ralph Scorza LLC has no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials. (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) as all materials and data are freely available.

The orphan nuclear receptor TLX is predominantly expressed in the central nervous system and is an important factor regulating the maintenance and self-renewal of neural stem cells from embryonic development through adulthood. In adolescence and adulthood, TLX expression is restricted to the neurogenic niches of the brain: the dentate gyrus of the hippocampus and the subventricular zone. The adolescent period is critical for maturation of the hippocampus with heightened levels of neurogenesis observed in rodents. Therefore, we investigated whether lentiviral silencing of TLX expression (TLX knockdown) in the dorsal dentate gyrus of juvenile rats incurred differential impairments in behaviour during late adolescence and adulthood. Our results showed that knockdown of TLX in the dorsal dentate gyrus led to a decrease in cell proliferation in the dorsal but not ventral dentate gyrus. At a behavioural level we observed differential effects in adolescence and adulthood across a number of parameters. A hyperactive phenotype was present in adolescent but not adult TLX knockdown rats, and an increase in immobility during adolescence and in swimming frequency during adulthood was observed in the forced swim test. There was an increased defecation frequency in the open field during adulthood but not adolescence. There were no changes in cognitive performance on hippocampus-dependent tasks or in anxiety-related behaviours. In conclusion, silencing of TLX in the dorsal dentate gyrus led to impairments in hippocampal-independent behaviours which either did not persist or were reversed during adulthood. The current data highlight the temporal importance and function of the nuclear receptor TLX during development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Patients with ovarian cancer commonly have a poor prognosis, owing to its invasiveness and distant metastasis. Studies have found TM4SF1 participates in regulating tumor cell invasion and migration. Therefore, it is expected to become a target for anti-invasion and metastasis in ovarian cancer., Methods: The expression of TM4SF1 in normal ovarian epithelial tissues, benign ovarian tumor tissues, primary foci of epithelial ovarian cancer and the matched lymph mode metastatic foci was detected using immunohistochemistry to analyze its association with prognosis. The expression of TM4SF1 in HO8910PM, SKOV3 was inhibited using RNAi, and the growth, proliferation, migration, invasion abilities of HO8910PM and SKOV3 cells and the growth of xenograft tumors in nude mice were examined., Results: (1) The positive expression rate of TM4SF1 protein in epithelial ovarian cancer tissues (90.90%) was higher than that in benign ovarian tumor tissues (65.22%) and normal ovarian epithelial tissues (31.25%), and both differences were significant (P < 0.05). The expression of TM4SF1 protein was positive in all metastatic lymph node foci and matched primary foci (100%). (2) The level of TM4SF1 protein expression was positively correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage and histological grade. However, The positive TM4SF1 protein expression was not an independent factor of prognosis (P > 0.05). (3) Silencing TM4SF1 expression did not affect growth, proliferation, or cell cycle distribution but inhibited the migration and invasion abilities of HO8910PM and SKOV3 cells. Silencing TM4SF1 expression inhibited the growth of xenograft tumors in nude mice., Conclusion: TM4SF1 is a potential target for anti-invasion and metastasis in ovarian cancer.

The genetic model organism, Caenorhabditis elegans (C. elegans), shares many genes with humans and is the best-annotated of the eukaryotic genome. Therefore, the identification of new genes and pathways is unlikely. Nevertheless, host-pathogen interaction studies from viruses, recently discovered in the environment, has created new opportunity to discover these pathways. For example, the exogenous RNAi response in C. elegans by the Orsay virus as seen in plants and other eukaryotes is not systemic and transgenerational, suggesting different RNAi pathways between these organisms. Using a bioinformatics meta-analysis approach, we show that the top 17 genes differentially-expressed during C. elegans infection by Orsay virus are functionally uncharacterized genes. Furthermore, functional annotation using similarity search and comparative modeling, was able to predict folds correctly, but could not assign easily function to the majority. However, we could identify gene expression studies that showed a similar pattern of gene expression related to toxicity, stress and immune response. Those results were strengthened using protein-protein interaction network analysis. This study shows that novel molecular pathway components, of viral innate immune response, can be identified and provides models that can be further used as a framework for experimental studies. Whether these features are reminiscent of an ancient mechanism evolutionarily conserved, or part of a novel pathway, remain to be established. These results reaffirm the tremendous value of this approach to broaden our understanding of viral immunity in C. elegans.

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and an ideal model organism for various studies. Chitin synthase (CHS) catalyses chitin synthesis, a critical structural component of insect exoskeletons. Here, we identified a CHS gene from A. pisum, ApisCHS. The ApisCHS expression profiles showed that ApisCHS was expressed in various developmental stages and in all tested tissues of A. pisum, including the epidermis, embryo, gut and haemolymph. Notably, ApisCHS exhibited peak expression in the middle of each nymphal period and was extremely highly expressed in the epidermis and embryo. RNA interference (RNAi) showed that ~600 ng of dsRNA is an effective dose for gene silencing by injection for dsRNA delivery; moreover, 1200 ng·μL -1 dsRNA induced CHS gene silencing by a plant-mediated feeding approach. A 44.7% mortality rate and a 51.3% moulting rate were observed 72 h after injection of dsApisCHS into fourth-instar nymphs, compared with the levels in the control (injected with dsGFP). Moreover, a longer period was required for nymph development and a 44.2% deformity rate among newborn nymphs was obtained upon ingestion of dsApisCHS. These results suggest that ApisCHS plays a critical role in nymphal growth and embryonic development in pea aphids, and is a potential target for RNAi-based aphid pest control.

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs. IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism., (Copyright © 2019 American Society for Microbiology.)

Mitoviruses (genus Mitovirus , family Narnaviridae ) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.2 to 4.4 kb with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase. Cryphonectria parasitica mitovirus 1 (CpMV1) from U.S. strain NB631 of the chestnut blight fungus, Cryphonectria parasitica , was the first virus identified as a mitochondrially replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single-conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriaceae, Cryphonectria radicalis and Valsa ceratosperma , also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae PCR-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and -deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, which is consistent with its mitochondrial replication. Collectively, the results of study provide a foundation to further explore the biology of mitoviruses. IMPORTANCE Capsidless mitoviruses, which are ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.2 to 4.4 kb, encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria parasitica mitovirus 1 (CpMV1), which was isolated from strain NB631 of the chestnut blight fungus ( Cryphonectria parasitica ), a model filamentous fungus for studying virus-host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica and different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and -competent strains showed clearly that the virus was unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus-host interactions., (Copyright © 2019 American Society for Microbiology.)

Both zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) are intimately involved in many aspects of early intestinal mucosal repair after acute injury, but the exact mechanisms that control their cellular abundances remain largely unknown. The present study shows that microRNA-222 (miR-222) interacts with the mRNAs encoding ZBP1 and PLCγ1 and regulates ZBP1 and PLCγ1 expression in intestinal epithelial cells (IECs). The biotinylated miR-222 bound specifically to the ZBP1 and PLCγ1 mRNAs in IECs. Ectopically expressed miR-222 precursor destabilized the ZBP1 and PLCγ1 mRNAs and consequently lowered the levels of cellular ZBP1 and PLCγ1 proteins. Conversely, decreasing the levels of cellular miR-222 by transfection with its antagonism increased the stability of the ZBP1 and PLCγ1 mRNAs and increased the levels of ZBP1 and PLCγ1 proteins. Overexpression of miR-222 also inhibited cell migration over the wounded area, which was partially abolished by overexpressing ZBP1 and PLCγ1. Furthermore, prevention of the increased levels of ZBP1 and PLCγ1 in the miR-222-silenced cells by transfection with specific small interfering RNAs targeting ZBP1 or PLCγ1 mRNA inhibited cell migration after wounding. These findings indicate that induced miR-222 represses expression of ZBP1 and PLCγ1 at the posttranscriptional level, thus inhibiting IEC migration during intestinal epithelial restitution after wounding.