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Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P < 0.05). Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). In the FMDV challenge study, one TGCS was completely protected, no clinical signs, no viremia and no viral RNA in the tissues, no non-structural antibody response, another one TGCS swine recovered after showing clinical signs for two days, whereas all of the normal control swine (NS) and Non-TGCS developed typical clinical signs, viremia and viral RNA was determined in the tissues, the non-structural antibody was determined, and one Non-TGCS swine died. The viral RNA load in the blood and tissues of the TGCS was reduced in both challenge doses. These results indicated that the TGCS displayed resistance to the FMDV infection. Immune cells, including CD3 + , CD4 + , CD8 + , CD21 + , and CD172 + cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine., (© 2021. The Author(s).)

The leaf curl disease of Jatropha caused by geminiviruses results in heavy economic losses. In the present study, we report the identification of a new strain of a Jatropha leaf curl Gujarat virus (JLCuGV), which encodes six ORFs with each one having RNA silencing suppressor activity. Therefore, three artificial microRNAs (amiRNAs; C1/C4, C2/C3 and V1/V2) were designed employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA and transformed in tobacco. The C1/C4 and C2/C3 amiRNA transgenics were resistant while V1/V2 amiRNA transgenics were tolerant against JLCuGV. The relative level of amiRNA inversely related to viral load indicating a correlation with disease resistance. The assessment of photosynthetic parameters suggests that the transgenics perform significantly better in response to JLCuGV infiltration as compared to wild type (WT). The metabolite contents were not altered remarkably in amiRNA transgenics, but sugar metabolism and tricarboxylic acid (TCA) cycle showed noticeable changes in WT on virus infiltration. The overall higher methylation and demethylation observed in amiRNA transgenics correlated with decreased JLCuGV accumulation. This study demonstrates that amiRNA transgenics showed enhanced resistance to JLCuGV while efficiently maintaining normalcy in their photosynthesis and metabolic pathways as well as homeostasis in the methylation patterns.

Trehalose-6-phosphate synthase (TPS) and trehalase (TRE) directly regulate trehalose metabolism and indirectly regulate chitin metabolism in insects. Real-time quantitative PCR (RT-qPCR) and RNA interference (RNAi) were used to detect the expressions and functions of the ApTPS and ApTRE genes. Abnormal phenotypes were found after RNAi of ApTRE in the Acyrthosiphon pisum. The molting deformities were observed in two color morphs, while wing deformities were only observed in the red morphs. The RNAi of ApTPS significantly down-regulated the expression of chitin metabolism-related genes, UDP-N-acetyglucosamine pyrophosphorylase (ApUAP), chitin synthase 2 (Apchs-2), Chitinase 2, 5 (ApCht2, 5), endo-beta-N-acetylglucosaminidase (ApENGase) and chitin deacetylase (ApCDA) genes at 24 h and 48 h; The RNAi of ApTRE significantly down-regulated the expression of ApUAP, ApCht1, 2, 8 and ApCDA at 24 h and 48 h, and up-regulated the expression of glucose-6-phosphate isomerase (ApGPI) and Knickkopf protein (ApKNK) genes at 48 h. The RNAi of ApTRE and ApTPS not only altered the expression of chitin metabolism-related genes but also decreased the content of chitin. These results demonstrated that ApTPS and ApTRE can regulate the chitin metabolism, deepen our understanding of the biological functions, and provide a foundation for better understanding the molecular mechanism of insect metamorphosis.

RNA viruses exist as populations of genome variants. Virus-infected plants accumulate 21-24 nucleotide small interfering RNAs (siRNAs) derived from viral RNA (virus-derived siRNAs) through gene silencing. This paper describes the profile of mutations in virus-derived siRNAs for three members of the family Potyviridae: Turnip mosaic virus (TuMV), Papaya ringspot virus (PRSV) and Wheat streak mosaic virus (WSMV). For TuMV in Arabidopsis thaliana, profiles were obtained for mechanically inoculated rosette leaves and systemically infected cauline leaves and inflorescence. Results are consistent with selection pressure on the viral genome imposed by local and systemic movement. By genetically removing gene silencing in the plant and silencing suppression in the virus, our results showed that antiviral gene silencing imposes selection in viral populations. Mutations in siRNAs derived from a PRSV coat protein transgene in the absence of virus replication showed the contribution of cellular RNA-dependent RNA polymerases to the generation of mutations in virus-derived siRNAs. Collectively, results are consistent with two sources of mutations in virus-derived siRNAs: viral RNA-dependent RNA polymerases responsible for virus replication and cellular RNA-dependent RNA polymerases responsible for gene silencing amplification.

Meloidogyne incognita is a plant-parasitic root-knot nematode (RKN, PPN) responsible for causing damage to several crops worldwide. In Caenorhabditis elegans, the DAF-16 and SKN-1 transcription factors (TFs) orchestrate aging, longevity, and defense responses to several stresses. Here, we report that MiDaf16-like1 and MiSkn1-like1, which are orthologous to DAF-16 and SKN-1 in C. elegans, and some of their targets, are modulated in M. incognita J2 during oxidative stress or plant parasitism. We used RNAi technology for the stable production of siRNAs in planta to downregulate the MiDaf16-like1 and MiSkn1-like1 genes of M. incognita during host plant parasitism. Arabidopsis thaliana and Nicotiana tabacum overexpressing a hairpin-derived dsRNA targeting these genes individually (single-gene silencing) or simultaneously (double-gene silencing) were generated. T 2 plants were challenged with M. incognita and the number of eggs, galls, and J2, and the nematode reproduction factor (NRF) were evaluated. Our data indicate that MiDaf16-like1, MiSkn1-like1 and some genes from their networks are modulated in M. incognita J2 during oxidative stress or plant parasitism. Transgenic A. thaliana and N. tabacum plants with single- or double-gene silencing showed significant reductions in the numbers of eggs, J2, and galls, and in NRF. Additionally, the double-gene silencing plants had the highest resistance level. Gene expression assays confirmed the downregulation of the MiDaf16-like1 and MiSkn1-like1 TFs and defense genes in their networks during nematode parasitism in the transgenic plants. All these findings demonstrate that these two TFs are potential targets for the development of biotechnological tools for nematode control and management in economically important crops.

Aphids are important agricultural pests causing major yield losses worldwide. Since aphids can rapidly develop resistance to chemical insecticides there is an urgent need to find alternative aphid pest management strategies. Despite the economic importance of bluegreen aphid (Acyrthosiphon kondoi), very few genetic resources are available to expand our current understanding and help find viable control solutions. An artificial diet is a desirable non-invasive tool to enable the functional characterisation of genes in bluegreen aphid and discover candidate target genes for future use in RNA interference (RNAi) mediated crop protection against aphids. To date no artificial diet has been developed for bluegreen aphid, so we set out to develop a suitable diet by testing and optimising existing diets. Here, we describe an artificial diet for rearing bluegreen aphid and also provide a proof of concept for the supplementation of the diet with RNAi molecules targeting the salivary gland transcript C002 and gap gene hunchback, resulting in bluegreen aphid mortality which has not yet been documented in this species. Managing this pest, for example via RNAi delivery through artificial feeding will be a major improvement to test bluegreen aphid candidate target genes for future pest control and gain significant insights into bluegreen aphid gene function.

We have previously developed efficient peptide-based nucleic acid delivery vectors PF14 and NF55, where we have shown that these vectors preferentially transfect lung tissue upon systemic administration with the nucleic acid. In the current work, we have explored the utilization and potential of these vectors for the lung-targeted gene therapy. Accordingly, we assessed the efficacy of these peptides in (i) two different lung disease models - acute lung inflammation and asthma in mice and (ii) using two different nucleic acid cargos - siRNA and pDNA encoding shRNA. Using RNAi against cytokine TNFα, we showed efficient anti-inflammatory effects in both disease models and observed decreased disease symptoms. Our results highlight the potential of our transfection vectors for lung gene therapy.

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 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.

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.

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.

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.

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 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.

Analysis of complex biological functions usually requires tissue-specific genetic manipulations in multicellular organisms. The C. elegans germline plays regulatory roles not only in reproduction, but also in metabolism, stress response and ageing. Previous studies have used mutants of rrf-1, which encodes an RNA-directed RNA polymerase, as a germline-specific RNAi tool. However, the rrf-1 mutants showed RNAi activities in somatic tissues. Here we constructed a germline-specific RNAi strain by combining an indel mutation of rde-1, which encodes an Argonaute protein that functions cell autonomously to ensure RNAi efficiency, and a single copy rde-1 transgene driven by the sun-1 germline-specific promoter. The germline RNAi efficiency and specificity are confirmed by RNAi phenocopy of known mutations, knockdown of GFP reporter expression, as well as quantitative RT-PCR measurement of tissue-specific mRNAs upon RNAi knockdown. The germline-specific RNAi strain shows no obvious deficiencies in reproduction, lipid accumulation, thermo-tolerance and life span compared to wild-type animals. By screening an RNAi sub-library of phosphatase genes, we identified novel regulators of thermo-tolerance. Together, we have created a useful tool that can facilitate the genetic analysis of germline-specific functions in C. elegans.

Gsdf is a key gene for testicular differentiation in teleost. However, little is known about the function of Gsdf in Chinese tongue sole (Cynoglossus semilaevis). In this study, we obtained the full-length Gsdf gene (CS-Gsdf), and functional characterization revealed its potential participation during germ cell differentiation in testes. CS-Gsdf transcription was predominantly detected in gonads, while the levels in testes were significantly higher than those in ovaries. During the different developmental stages in male gonads, the mRNA level was significantly upregulated at 86 dph, and a peak appeared at 120 dph; then, the level decreased at 1 and 2 yph. In situ hybridization revealed that CS-Gsdf mRNA was mainly localized in the Sertoli cells, spermatogonia, and spermatids in mature testes. After CS-Gsdf knockdown in the male testes cell line by RNA interference, a series of sex-related genes was influenced, including several sex differentiation genes, CS-Wnt4a, CS-Cyp19a1a and CS-Star. Based on these data, we speculated that CS-Gsdf may play a positive role in germ differentiation and proliferation via influencing genes related to sex differentiation.

RNA-induced silencing complex (RISC) plays a critical role in small interfering RNA (siRNA) and microRNAs (miRNA) pathways. Accumulating evidence has demonstrated that the major RISC members (AGO, DICER, TRBP, PACT and GW182) represent expression discrepancies or multiple orthologues/paralogues in different species. To elucidate their evolutionary characteristics, an integrated evolutionary analysis was performed. Here, animal and plant AGOs were divided into three classes (multifunctional AGOs, siRNA-associated AGOs and piRNA-associated AGOs for animal AGOs and multifunctional AGOs, siRNA-associated AGOs and complementary functioning AGOs for plant AGOs). Animal and plant DICERs were grouped into one class (multifunctional DICERs) and two classes (multifunctional DICERs and siRNA-associated DICERs), respectively. Protista/fungi AGOs or DICERs were specifically associated with the siRNA pathway. Additionally, TRBP/PACT/GW182 were identified only in animals, and all of them functioned in the miRNA pathway. Mammalian AGOs, animal DICERs and chordate TRBP/PACT were found to be monophyletic. A large number of gene duplications were identified in AGO and DICER groups. Taken together, we provide a comprehensive evolutionary analysis, describe a phylogenetic tree-based classification of the major RISC members and quantify their gene duplication events. These findings are potentially useful for classifying RISCs, optimizing species-specific RISCs and developing research model organisms.

We investigated the functional role of CEACAM1 in a spontaneous metastasis xenograft model of human melanoma in scid mice using BRAF wildtype MeWo cells with and without RNAi mediated knockdown of CEACAM1. Tumors from the xenograft model were subjected to whole genome expression analysis and metastasis was quantified histologically. Results and identified markers were verified using tissue samples of over 100 melanoma patients. Knockdown of CEACAM1 prolonged the animals' survival by significantly reducing subcutaneous growth of MeWo tumors and spontaneous lung metastasis. Microarray analysis revealed a strong influence of CEACAM1 knockdown on the network of EMT associated genes in the xenograft tumors (e.g. downregulation of BRAF, FOSL1, NRAS and TWIST). IGFBP7 and Latexin (highest up- and downregulated expression in microarray analysis) were found to be associated with longer and shorter survival, respectively, of melanoma patients. High FOSL1 and altered TWIST1 expression were found to be correlated with shortened survival in the cohort of melanoma patients. After a stepwise selection procedure combining above markers, multivariate analysis revealed IGFBP7, Latexin and altered TWIST to be prognostic markers for death. CEACAM1 could be a target for melanoma therapy as an alternative to (or in combination with) immune checkpoint and BRAF inhibitors.

Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.

The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified 18S rRNA and 60S RP as the best genes to use as a reference in reverse-transcriptase quantitative PCR (RT-qPCR). Homologs of 13 genes that were shown to function as effective RNAi targets in other insects were identified and evaluated by injecting dsRNA targeting these homologs into BMSB adults. Five out of 13 dsRNAs tested caused more than 70% mortality by seven days after injection of dsRNA. Feeding dsRNA targeting five of these genes (IAP, ATPase, SNF7, GPCR, and PPI) to nymphs caused more than 70% mortality by three of the five dsRNAs tested. These data suggest that feeding dsRNA causes target gene knockdown and mortality in BMSB.

The MADS-box transcription factors play essential roles in many physiological and biochemical processes of plants, especially in fruit ripening. Here, a tomato MADS-box gene, SlCMB1, was isolated. SlCMB1 expression declined with the fruit ripening from immature green to B + 7 (7 days after Breaker) fruits in the wild type (WT) and was lower in Nr and rin mutants fruits. Tomato plants with reduced SlCMB1 mRNA displayed delayed fruit ripening, reduced ethylene production and carotenoid accumulation. The ethylene production in SlCMB1-RNAi fruits decreased by approximately 50% as compared to WT. The transcripts of ethylene biosynthesis genes (ACS2, ACS4, ACO1 and ACO3), ethylene-responsive genes (E4, E8 and ERF1) and fruit ripening-related genes (RIN, TAGL1, FUL1, FUL2, LoxC and PE) were inhibited in SlCMB1-RNAi fruits. The carotenoid accumulation was decreased and two carotenoid synthesis-related genes (PSY1 and PDS) were down-regulated while three lycopene cyclase genes (CYCB, LCYB and LCYE) were up-regulated in transgenic fruits. Furthermore, yeast two-hybrid assay showed that SlCMB1 could interact with SlMADS-RIN, SlMADS1, SlAP2a and TAGL1, respectively. Collectively, these results indicate that SlCMB1 is a new component to the current model of regulatory network that regulates ethylene biosynthesis and carotenoid accumulation during fruit ripening.

Panonychus citri is one of the most damaging pests of horticultural crops. Conventional control of this pest population through pesticides has led to the enhanced pest resistance. Management of P. citri population through RNAi, is still largely unknown. In oviparous organisms, fabrication and development of yolk protein play a vital role in the reproduction. Vitellin (Vn) is the source of eggs storage that helps in proper functioning of Vitellogenin (Vg) and Vitellogenin receptor (VgR). VgR is very compulsory protein for the development of Vg into oocytes. In the current study, Vg (PcVg) and VgR (PcVgR) genes were studied and their expressions at different developmental stages were quantified by RT-qPCR. Females treated with dsRNA of PcVg and PcVgR genes exhibited reduction in gene expression. Down regulation of target genes significantly effected oviposition and reduced the egg laying capacity up to 48% as compared to control (ds-egfp). Synergistic effect of target gene's dsRNA was also accessed that reduced the egg laying up to 60.42%. Furthermore, combination of target dsRNA on deutonymph and protonymph also resulted in 67% and 70% reduction in eggs, respectively. Synergistic effect of dsRNA at 1000 ng/ul resulted in longer life span as compared to control treatments. This study suggests to develop a new strategy of P. citri population control by reducing its reproduction.

High CD44 expression is associated with enhanced malignant potential in esophageal squamous cell carcinoma (ESCC), among the deadliest of all human carcinomas. Although alterations in autophagy and CD44 expression are associated with poor patient outcomes in various cancer types, the relationship between autophagy and cells with high CD44 expression remains incompletely understood. In transformed oesophageal keratinocytes, CD44 Low -CD24 High (CD44L) cells give rise to CD44 High -CD24 -/Low (CD44H) cells via epithelial-mesenchymal transition (EMT) in response to transforming growth factor (TGF)-β. We couple patient samples and xenotransplantation studies with this tractable in vitro system of CD44L to CD44H cell conversion to investigate the functional role of autophagy in generation of cells with high CD44 expression. We report that high expression of the autophagy marker cleaved LC3 expression correlates with poor clinical outcome in ESCC. In ESCC xenograft tumours, pharmacological autophagy inhibition with chloroquine derivatives depletes cells with high CD44 expression while promoting oxidative stress. Autophagic flux impairment during EMT-mediated CD44L to CD44H cell conversion in vitro induces mitochondrial dysfunction, oxidative stress and cell death. During CD44H cell generation, transformed keratinocytes display evidence of mitophagy, including mitochondrial fragmentation, decreased mitochondrial content and mitochondrial translocation of Parkin, essential in mitophagy. RNA interference-mediated Parkin depletion attenuates CD44H cell generation. These data suggest that autophagy facilitates EMT-mediated CD44H generation via modulation of redox homeostasis and Parkin-dependent mitochondrial clearance. This is the first report to implicate mitophagy in regulation of tumour cells with high CD44 expression, representing a potential novel therapeutic avenue in cancers where EMT and CD44H cells have been implicated, including ESCC.

Aberrant Hedgehog/GLI signaling has been implicated in a diverse spectrum of human cancers, but its role in lung adenocarcinoma (LAC) is still under debate. We show that the downstream effector of the Hedgehog pathway, GLI1, is expressed in 76% of LACs, but in roughly half of these tumors, the canonical pathway activator, Smoothened, is expressed at low levels, possibly owing to epigenetic silencing. In LAC cells including the cancer stem cell compartment, we show that GLI1 is activated noncanonically by MAPK/ERK signaling. Different mechanisms can trigger the MAPK/ERK/GLI1 cascade including KRAS mutation and stimulation of NRP2 by VEGF produced by the cancer cells themselves in an autocrine loop or by stromal cells as paracrine cross talk. Suppression of GLI1, by silencing or drug-mediated, inhibits LAC cells proliferation, attenuates their stemness and increases their susceptibility to apoptosis in vitro and in vivo. These findings provide insight into the growth of LACs and point to GLI1 as a downstream effector for oncogenic pathways. Thus, strategies involving direct inhibition of GLI1 may be useful in the treatment of LACs.

RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA.

The RNA interference (RNAi) plays a critical role in gene regulation in a variety of eukaryotic organisms. However, the role of RNAi remains largely unclear in plant pathogenic fungi. In this study, we explored the roles of core components of the RNAi pathway in Fusarium graminearum, the major causal agent of wheat head blight. Our results demonstrated that the hairpin RNA (hpRNA) can efficiently silence the expression level of target gene, and the argonaute protein FgAgo1 and dicer protein FgDicer2 are important in this silencing process. RNAi machinery was not involved in growth, abiotic stress and pathogenesis in F. graminearum under tested conditions. We firstly applied high-throughput sequencing technology to elucidate small RNA (17-40 nucleotides) (sRNA) transcriptome in F. graminearum, and found that a total of forty-nine micro-like-RNA (milRNA) candidates were identified in the wild-type and ∆FgDICER2, and twenty-four of them were FgDicer2-dependent. Fg-milRNA-4 negatively regulated expression of its target gene. Taken together, our results indicated that the hpRNA-induced gene silencing was a valuable genetic tool for exploring gene function in F. graminearum. FgAgo1 and FgDicer2 proteins played a critical role in the hpRNA mediated gene silencing process. In addition, FgDicer2 was involved in sRNA transcription and milRNA generation in this fungus.

Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

During Drosophila metamorphosis, the single-cell layer of fat body tissues gradually dissociates into individual cells. Via a fat body-specific RNAi screen in this study, we found that two matrix metalloproteinases (MMPs), Mmp1 and Mmp2, are both required for fat body cell dissociation. As revealed through a series of cellular, biochemical, molecular, and genetic experiments, Mmp1 preferentially cleaves DE-cadherin-mediated cell-cell junctions, while Mmp2 preferentially degrades basement membrane (BM) components and thus destroy cell-BM junctions, resulting in the complete dissociation of the entire fat body tissues into individual cells. Moreover, several genetic interaction experiments demonstrated that the roles of Mmp1 and Mmp2 in this developmental process are cooperative. In conclusion, Mmp1 and Mmp2 induce fat body cell dissociation during Drosophila metamorphosis in a cooperative yet distinct manner, a finding that sheds light on the general mechanisms by which MMPs regulate tissue remodeling in animals.

RNA interference can induce heritable gene silencing, but it remains unexplored whether similar mechanisms play a general role in responses to cues that occur in the wild. We show that transient, mild heat stress in the nematode Caenorhabditis elegans results in changes in messenger RNA levels that last for more than one generation. The affected transcripts are enriched for genes targeted by germline siRNAs downstream of the piRNA pathway, and worms defective for germline RNAi are defective for these heritable effects. Our results demonstrate that a specific siRNA pathway transmits information about variable environmental conditions between generations.

RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.

The Forkhead Box M1 (FOXM1) transcription factor has been considered as a potential target for the prevention and/or therapeutic intervention in human carcinomas because of its roles in tumorigenesis and tumor progression through regulating the expression of genes relevant to cell proliferation and transformation. In this study, FOXM1 was found to express strongly in both clinical tissue specimens and human hepatocellular carcinoma (HCC) cell lines such as Huh-6, Huh-7 and HepG2. The knockdown of FOXM1 expression through an adenovirus vector (named AdFOXM1shRNA), which expresses a short hairpin RNA to downregulate FOXM1 expression specifically, diminished the proliferation of Huh-7 and HepG2 cells and anchorage-independent growth of Huh-7 cells. Furthermore, we assessed the efficacy of AdFOXM1shRNA for tumor gene therapy with the Huh-7 cell xenograft mouse model and found that the tumor growth was significantly suppressed when inoculated mice were injected with AdFOXM1shRNA in the tumors. Together, our results suggest that FOXM1 is a potential therapeutic target for HCC and AdFOXM1shRNA may be an additional gene therapeutic intervention for HCC treatment.

Intraspecific male-male aggression, which is important for sexual selection, is regulated by environment, experience and internal states through largely undefined molecular and cellular mechanisms. To understand the basic neural pathway underlying the modulation of this innate behavior, we established a behavioral assay in Drosophila melanogaster and investigated the relationship between sexual experience and aggression. In the presence of mating partners, adult male flies exhibited elevated levels of aggression, which was largely suppressed by prior exposure to females via a sexually dimorphic neural mechanism. The suppression involved the ability of male flies to detect females by contact chemosensation through the pheromone-sensing ion channel ppk29 and was mediated by male-specific GABAergic neurons acting on the GABAA receptor RDL in target cells. Silencing or activating this circuit led to dis-inhibition or elimination of sex-related aggression, respectively. We propose that the GABAergic inhibition represents a critical cellular mechanism that enables prior experience to modulate aggression.

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

Levels of eukaryotic initiation factor 4E (eIF4E) are frequently elevated in human cancers and in some instances have been associated with poor prognosis and outcome. Here we utilize transgenic and allograft breast cancer models to demonstrate that increased mammalian target of rapamycin (mTOR) signalling can be a significant contributor to breast cancer progression in vivo. Suppressing mTOR activity, as well as levels and activity of the downstream translation regulators, eIF4E and eIF4A, delayed breast cancer progression, onset of associated pulmonary metastasis in vivo and breast cancer cell invasion and migration in vitro. Translation of vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP9) and cyclin D1 mRNAs, which encode products associated with the metastatic phenotype, is inhibited upon eIF4E suppression. Our results indicate that the mTOR/eIF4F axis is an important contributor to tumor maintenance and progression programs in breast cancer. Targeting this pathway may be of therapeutic benefit.

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-β interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-β pathway and implicate this mechanism in the maintenance of genome integrity.

Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial responses to axon injury is molecularly encoded by unique isoforms of the Drosophila melanogaster engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a previously undescribed immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II-Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates that a balance of opposing Draper-I and Draper-II signaling events is essential to maintain glial sensitivity to brain injury.

Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca(2+)-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca(2+)-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle-associated SNAREs.

The protein kinase calcium/calmodulin-dependent kinase II (CaMKII) predominantly consists of the α and β isoforms in the brain. Although CaMKIIα functions have been elucidated, the isoform-specific catalytic functions of CaMKIIβ have remained unknown. Using knockdown analyses in primary rat neurons and in the rat cerebellar cortex in vivo, we report that CaMKIIβ operates at the centrosome in a CaMKIIα-independent manner to drive dendrite retraction and pruning. We also find that the targeting protein PCM1 (pericentriolar material 1) localizes CaMKIIβ to the centrosome. Finally, we uncover the E3 ubiquitin ligase Cdc20-APC (cell division cycle 20-anaphase promoting complex) as a centrosomal substrate of CaMKIIβ. CaMKIIβ phosphorylates Cdc20 at Ser51, which induces Cdc20 dispersion from the centrosome, thereby inhibiting centrosomal Cdc20-APC activity and triggering the transition from growth to retraction of dendrites. Our findings define a new, isoform-specific function for CaMKIIβ that regulates ubiquitin signaling at the centrosome and thereby orchestrates dendrite patterning, with important implications for neuronal connectivity in the brain.

Transcription of immediate early genes (IEGs) in neurons is highly sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We found that several IEGs, such as Arc (also known as Arg3.1), are poised for near-instantaneous transcription by the stalling of RNA polymerase II (Pol II) just downstream of the transcription start site in rat neurons. Depletion through RNA interference of negative elongation factor, a mediator of Pol II stalling, reduced the Pol II occupancy of the Arc promoter and compromised the rapid induction of Arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that were expressed later and largely lacked promoter-proximal Pol II stalling. Together, our data strongly indicate that the rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory.

DNA methylation regulates gene transcription and has been suggested to encode psychopathologies derived from early life stress. We found that methylation regulated the expression of the Crf (also known as Crh) gene and that chronic social stress in adult mice induced long-term demethylation of this genomic region. Demethylation was observed only in the subset of defeated mice that displayed social avoidance and site-specific knockdown of Crf attenuated the stress-induced social avoidance.

Although muscarinic acetylcholine receptors (mAChRs) and NMDA receptors (NMDARs) are important for synaptic plasticity, learning and memory, the manner in which they interact is poorly understood. We found that stimulation of muscarinic receptors, either by an agonist or by the synaptic release of acetylcholine, led to long-term depression (LTD) of NMDAR-mediated synaptic transmission. This form of LTD involved the release of Ca2+ from IP₃-sensitive intracellular stores and was expressed via the internalization of NMDARs. Our results suggest that the molecular mechanism involves a dynamic interaction between the neuronal calcium sensor protein hippocalcin, the clathrin adaptor molecule AP2, the postsynaptic density enriched protein PSD-95 and NMDARs. We propose that hippocalcin binds to the SH3 region of PSD-95 under basal conditions, but it translocates to the plasma membrane on sensing Ca2+; in doing so, it causes PSD-95 to dissociate from NMDARs, permitting AP2 to bind and initiate their dynamin-dependent endocytosis.

Trafficking of AMPA receptors (AMPARs) is important for many forms of synaptic plasticity. However, the link between activity and resulting synaptic alterations is not fully understood. We identified a direct interaction between N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase involved in membrane fusion events and stabilization of surface AMPARs, and Polo-like kinase- 2 (Plk2), an activity-inducible kinase that homeostatically decreases excitatory synapse number and strength. Plk2 disrupted the interaction of NSF with the GluA2 subunit of AMPARs, promoting extensive loss of surface GluA2 in rat hippocampal neurons, greater association of GluA2 with adaptor proteins PICK1 and GRIP1, and decreased synaptic AMPAR current. Plk2 engagement of NSF, but not Plk2 kinase activity, was required for this mechanism and occurred through a motif in the Plk2 protein that was independent of the canonical polo box interaction sites. These data reveal that heightened synaptic activity, acting through Plk2, leads to homeostatic decreases in surface AMPAR expression via the direct dissociation of NSF from GluA2.

Brain structure and size require precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, and mechanistic explanations of how aberrant NSC division causes the reduced brain size seen in microcephaly are lacking. Here we show that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly because of INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes, as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number and genomic stability. In utero rescue experiments showed that a key function of Magoh is to control levels of the microcephaly-associated protein Lis1 during neurogenesis. Our results uncover requirements for the EJC in brain development, NSC maintenance and mitosis, thereby implicating this complex in the pathogenesis of microcephaly.

Understanding the fundamental role of soluble NSF attachment protein receptor (SNARE) complexes in membrane fusion requires knowledge of the spatiotemporal dynamics of their assembly. We visualized complexin (cplx), a cytosolic protein that binds assembled SNARE complexes, during single exocytic events in live cells. We found that cplx appeared briefly during full fusion. However, a truncated version of cplx containing only the SNARE-complex binding region persisted at fusion sites for seconds and caused fusion to be transient. Resealing pores with the mutant cplx only partially released transmitter and lipid probes, indicating that the pores are narrow and not purely lipidic in structure. Depletion of cplx similarly caused secretory cargo to be retained. These data suggest that cplx is recruited at a late step in exocytosis and modulates fusion pores composed of SNARE complexes.

Ceramide can be converted into sphingomyelin by sphingomyelin synthases (SMS) 1 and 2. In this study, we show that in human leukemia Jurkat cells, which express mainly SMS1, Fas ligand (FasL) treatment inhibited SMS activity in a dose- and time-dependent manner before nuclear fragmentation. The SMS inhibition elicited by FasL (1) was abrogated by benzyloxycarbonyl valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor; (2) did not occur in caspase-8-deficient cells and (3) was not affected in caspase-9-deficient cells. Western blot experiments showed SMS1 cleavage in a caspase-dependent manner upon FasL treatment. In a cell-free system, caspase-2, -7, -8 and -9, but not caspase-3 and -10, cleaved SMS1. In HeLa cells, SMS1 was Golgi localized and relocated throughout the cytoplasm in cells exhibiting an early apoptotic phenotype on FasL treatment. zVAD-fmk prevented FasL-induced SMS1 relocation. Thus, FasL-mediated SMS1 inhibition and relocation depend on caspase activation and likely represent proximal events in Fas signaling. FasL-induced ceramide production and cell death were enhanced in cells stably expressing an siRNA against SMS1. Conversely, in cells stably overexpressing SMS1, FasL neither increased ceramide generation nor efficiently induced cell death. Altogether, our data show that SMS1 is a novel caspase target that is functionally involved in the regulation of FasL-induced apoptosis.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a main responder to intracellular hypoxia and is overexpressed in many human cancers, including renal cell carcinoma (RCC). To better understanding of the role of HIF-1alpha in the tumorigenicity of RCC, we used short-hairpin RNA (shRNA) interference to inhibit HIF-1alpha expression in the human renal cancer cell line, Caki-1 and OS-RC-2. Silencing of HIF-1alpha significantly reduced the expression of HIF-1alpha in both of renal cancer cell lines. In vitro downregulation of HIF-1alpha inhibited Caki-1 and OS-RC-2 cell growth, migration and invasion. The results further showed that HIF-1alpha silencing resulted in caspase-dependent apoptosis of Caki-1 and OS-RC-2 through regulation of PI3K/Akt pathway and Bcl-2-related proteins expression. In vivo animal studies showed that tumor growth was significantly inhibited in HIF-1alpha shRNA-transfected RCC. Intratumor gene therapy with polyethylenimine-loaded HIF-1alpha shRNA also resulted in tumor growth suppression. Thus, this study demonstrates that downregulation of HIF-1alpha could suppress tumorigenicity of RCC through induction of apoptosis, and HIF-1alpha shRNA may be a promising strategy for the treatment of RCC.

ApoJ/Clusterin (CLU) is a heterodimeric protein localized in the nucleus, cytoplasm or secretory organelles and involved in cell survival and neoplastic transformation. Its function in human cancer is still highly controversial. In this study, we examined the prostate of mice in which CLU has been genetically inactivated. Surprisingly, we observed transformation of the prostate epithelium in the majority of CLU knockout mice. Either PIN (prostate intraepithelial neoplasia) or differentiated carcinoma was observed in 100 and 87% of mice with homozygous or heterozygous deletion of CLU, respectively. Crossing CLU knockout with TRAMP (prostate cancer prone) mice results in a strong enhancement of metastatic spread. Finally, CLU depletion causes tumourigenesis in female TRAMP mice, which are normally cancer free. Mechanistically, deletion of CLU induces activation of nuclear factor-kB, a potentially oncogenic transcription factor important for the proliferation and survival of prostate cells.