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Drosophila photoreceptor cells (PRCs) are highly polarized epithelial cells. Their apical membrane is further subdivided into the stalk membrane and the light-sensing rhabdomere. The photo-pigment Rhodopsin1 (Rh1) localizes to the rhabdomere, whereas the apical determinant Crumbs (Crb) is enriched at the stalk membrane. The proteoglycan Eyes shut (Eys) is secreted through the apical membrane into an inter-rhabdomeral space. Rh1, Crb, and Eys are essential for PRC development, normal vision, and PRC survival. Human orthologs of all three proteins have been linked to retinal degenerative diseases. Here, we describe an RNAi-based screen examining the importance of approximately 240 trafficking-related genes in apical trafficking of Eys, Rh1, and Crb. We found 28 genes that have an effect on the localization and/or levels of these apical proteins and analyzed several factors in more detail. We show that the Arf GEF protein Sec71 is required for biosynthetic traffic of both apical and basolateral proteins, that the exocyst complex and the microtubule-based motor proteins dynein and kinesin promote the secretion of Eys and Rh1, and that Syntaxin 7/Avalanche controls the endocytosis of Rh1, Eys, and Crb.Article summeryPhotoreceptor cells (PRCs) rely on polarized vesicle trafficking to deliver key secreted and transmembrane proteins to their correct locations. Failure to do so causes defects in PRC development, function, and survival leading to retinal disease. Using the fruit fly Drosophila as a model we have identified 28 genes that are required for the trafficking of the three apical proteins Rhodopsin 1, Crumbs, and Eyes Shut. Human homologs of all three genes are associated with retinal degeneration. We characterized several genes to reveal novel mechanisms of vesicle trafficking in photoreceptor cells at different points in the biosynthetic or endocytotic pathways.

Contains fulltext : 202928.pdf (Publisher’s version ) (Open Access) Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants.

Most gene functions were detected by screens in very few model organisms but it has remained unclear how comprehensive these data are. Here, we expanded our RNAi screen in the red flour beetleTribolium castaneumto cover more than half of the protein-coding genes and we compared the gene sets involved in several processes between beetle and fly.We find that around 50 % of the gene functions are detected in both species while the rest was found only in fly (~10%) or beetle (~40%) reflecting both technical and biological differences. We conclude that work in complementary model systems is required to gain a comprehensive picture on gene functions documented by the annotation of novel GO terms for 96 genes studied here. The RNAi screening resources developed in this project, the expanding transgenic tool-kit and our large-scale functional data makeT. castaneuman excellent model system in that endeavor.

The ability of the immune system to avoid autoimmune disease relies on tolerization of thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, some of which belong to the 3’end processing complex, suggesting they might impact transcript stability and levels through an effect on 3’UTR shortening. We discovered that Aire-sensitive genes display a pronounced preference for short-3’UTR transcript isoforms in mTECs, a feature preceding Aire’s expression and correlated with the preferential selection of proximal polyA sites by the 3’end processing complex. Through an RNAi screen and generation of a lentigenic mouse, we found that one factor, Clp1, promotes 3’UTR shortening associated with higher transcript stability and expression of Aire-sensitive genes, revealing a post-transcriptional level of control of Aire-activated expression in mTECs.

Metabolic state is a potent modulator of sleep and circadian behavior, and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Across phyla, hormones secreted from adipose tissue act in the brain to control neural physiology and behavior to modulate sleep and metabolic state. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, we performed an RNAi screen selectively knocking down genes in the fat body. We found that knockdown of Phosphoribosylformylglycinamidine synthase/Pfas (Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes.

TRIM-NHL proteins are highly conserved regulators of developmental pathways in vertebrates and invertebrates. The TRIM-NHL family member NHL-2 in Caenorhabditis elegans functions as a miRNA cofactor to regulate developmental timing. Similar regulatory roles have been reported in other model systems, with the mammalian ortholog in mice, TRIM32, contributing to muscle and neuronal cell proliferation via miRNA activity. Given the interest associated with TRIM-NHL family proteins, we aimed to further investigate the role of NHL-2 in C. elegans development by using a synthetic RNAi screening approach. Using the ORFeome library, we knocked down 11,942 genes in wild-type animals and nhl-2 null mutants. In total, we identified 42 genes that produced strong reproductive synthetic phenotypes when knocked down in nhl-2 null mutants, with little or no change when knocked down in wild-type animals. These included genes associated with transcriptional processes, chromosomal integrity, and key cofactors of the germline small 22G RNA pathway.

Summary Molecular mechanisms responsible for the development of human skin epithelial cells are incompletely understood. As a consequence, the efficiency to establish a pure skin epithelial cell population from human induced pluripotent stem cells (hiPSCs) remains poor. Using an approach including RNAi and high-throughput imaging of early epithelial cells, we identified candidate kinases involved in their differentiation from hiPSCs. Among these, we found HIPK4 to be an important inhibitor of this process. Indeed, its silencing increased the amount of generated skin epithelial precursors at an early time point, increased the amount of generated keratinocytes at a later time point, and improved growth and differentiation of organotypic cultures, allowing for the formation of a denser basal layer and stratification with the expression of several keratins. Our data bring substantial input regarding regulation of human skin epithelial differentiation and for improving differentiation protocols from pluripotent stem cells., Highlights • High-throughput RNAi screen setup during human skin epithelial differentiation • Identification of HIPK4 as a crucial blocker of human skin epithelial differentiation • Improvement of human organotypic epithelial cultures after HIPK4 silencing, In this article, Larribère and colleagues establish a high-throughput RNAi screen on induced pluripotent stem cell-derived epithelial cells. This screen led to the identification of HIPK4 as a crucial blocker of human skin epithelial differentiation. Indeed, HIPK4 silencing increased the amount of early generated skin epithelial precursors, increased the amount of generated keratinocytes, and improved the differentiation of organotypic cultures.

In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon‐β (IFNB1) induction. Using a comprehensive microscopy‐based high‐content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A‐interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF‐κB p65 upon Sendai virus (SeV) infection. Notably, importin β1 (IMPβ1) knockdown—a hub protein highly targeted by several viruses—decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus‐mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPβ1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPβ1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF‐κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad‐spectrum antiviral therapies., Hepatitis C virus (HCV) hijacks the cellular host machinery to promote replication and to evade immune response. Using a microscopy‐based High Content Screening (HCS) assay, we demonstrated that HCV‐host interactors involved in nuclear transport are crucial for IRF3 and NFκB‐p65 signaling of IFNB1 response. To subvert innate immunity, NS3/4A interacts and cleaves IMPβ1, a key nucleocytoplasmic transport receptor, to prevent nuclear translocation of IRF3 and NFκB‐p65 transcription factors. Future studies will confirm if IMPβ1 is a prime target for several viruses to evade host defense.

International audience; Therapeutic resistance is a major clinical challenge in oncology. Evidence identifies cancer stem cells (CSCs) as a driver of tumor evolution. Accordingly, the key stemness property unique to CSCs may represent a reservoir of therapeutic target to improve cancer treatment. Here, we carried out a genome-wide RNA interference screen to identify genes that regulate breast CSCs-fate (bCSC). Using an interactome/regulome analysis, we integrated screen results in a functional mapping of the CSC-related processes. This network analysis uncovered potential therapeutic targets controlling bCSC-fate. We tested a panel of 15 compounds targeting these regulators. We showed that mifepristone, salinomycin, and JQ1 represent the best anti-bCSC activity. A combination assay revealed a synergistic interaction of salinomycin/JQ1 association to deplete the bCSC population. Treatment of primary breast cancer xenografts with this combination reduced the tumor-initiating cell population and limited metastatic development. The clinical relevance of our findings was reinforced by an association between the expression of the bCSC-related networks and patient prognosis. Targeting bCSCs with salinomycin/JQ1 combination provides the basis for a new therapeutic approach in the treatment of breast cancer.

Context-dependent changes in genetic interactions are an important feature of cellular pathways and their varying responses under different environmental conditions. However, methodological frameworks to investigate the plasticity of genetic interaction networks over time or in response to external stresses are largely lacking. To analyze the plasticity of genetic interactions, we performed a combinatorial RNAi screen in Drosophila cells at multiple time points and after pharmacological inhibition of Ras signaling activity. Using an image-based morphology assay to capture a broad range of phenotypes, we assessed the effect of 12768 pairwise RNAi perturbations in six different conditions. We found that genetic interactions form in different trajectories and developed an algorithm, termed MODIFI, to analyze how genetic interactions rewire over time. Using this framework, we identified more statistically significant interactions compared to end-point assays and further observed several examples of context-dependent crosstalk between signaling pathways such as an interaction between Ras and Rel which is dependent on MEK activity. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., eLife digest Within a cell, communication routes that involve many different genes work to control how the cell responds to the environment. Although different communication routes – so called signaling pathways – control different cell activities, they do not work in isolation. Instead, they form part of larger regulatory networks that maintain the cell in an appropriate state. As such, changing the activity of one pathway may in turn affect another seemingly unrelated pathway. The Ras signaling pathway helps to control when cells divide. When this signaling is not regulated correctly, cells can start to divide uncontrollably, leading to cancer. Drugs that suppress the activity of overactive Ras pathways could help to treat cancer. But how do the wider regulatory networks in the cell rewire themselves over time in response to this treatment? To investigate this question, Heigwer et al. used a method called RNA interference to alter the activity of different pairs of 168 genes in fruit fly cells that had been grown in the laboratory. This meant 12,768 gene interactions were examined in total. Some of the cells had been treated with a drug that suppresses Ras signaling. By developing a new cell imaging and analysis system, Heigwer et al. could examine how the cell’s regulatory networks were affected by the drug at three different time points after treatment. The results show that housekeeping genes, which handle basic cell duties, take more time to rewire their interactions than signaling pathways. Heigwer et al. also developed a computational method – called MODIFI – to analyze how environment and time affect how genes interact. This highlighted a number of signaling pathways that are strongly affected by the suppression of Ras signaling, including an unexpected immune signaling pathway. In the future, more research will be needed to study the context-dependency of interactions between genetic networks in different cell types and in living organisms. A better understanding of this context-dependency will be important to understand how cancerous cells develop drug resistance. The data collected by Heigwer et al. could also be used by other researchers to explain any unexpected gene interactions that affect the signaling pathways they are studying.

Stem cells are regulated both intrinsically and externally, including by signals from the local environment and distant organs. To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. Some genes, for example, versatile (vers), encoding a heterochromatin protein, regulates GSC fates differentially in different cell types and through multiple pathways. We also find that mitosis/cytokinesis proteins are especially important for male GSC maintenance. Our findings provide valuable insights and resources for studying stem cell regulation at the organismal level., Both intrinsic and external signals regulate stem cell fate. Here, the authors perform a genome-wide RNAi screen to identify regulators of testis germline stem cells (GSC) in Drosophila, finding more than half of the genes were external regulators and heterochromatin/cytokinesis proteins mediated GSC fate.

Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens invivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.

Although muscle development has been widely studied inDrosophila melanogasterthere are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle,Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered ~8,500 and ~5,000 genes, respectively, of a total of ~16,500 genes of theTriboliumgenome. Apart from many genes known fromDrosophilaas regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by theDrosophilaandTriboliumscreening approaches.

SummaryWe combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization, and “protein-level dependency” studies—a systematic technique that uncovers post-transcriptional regulation—to delineate the network of factors that control the expression of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs versus embryonic stem cells (ESCs). Through multiparametric data analyses, we predict that Tox4 is associating with the Paf1C complex, which maintains cell identity in both cell types, and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a framework for better understanding pluripotency and for dissecting the molecular events that govern the transition from the pre-implantation to the post-implantation state.

Stem cells present unique regenerative abilities, offering great potential for treatment of prevalent pathologies such as diabetes, neurodegenerative and heart diseases. Various research groups dedicated significant effort to identify sets of genes-so-called stemness signatures-considered essential to define stem cells. However, their usage has been hindered by the lack of comprehensive resources and easy-to-use tools. For this we developed StemChecker, a novel stemness analysis tool, based on the curation of nearly fifty published stemness signatures defined by gene expression, RNAi screens, Transcription Factor (TF) binding sites, literature reviews and computational approaches. StemChecker allows researchers to explore the presence of stemness signatures in user-defined gene sets, without carrying-out lengthy literature curation or data processing. To assist in exploring underlying regulatory mechanisms, we collected over 80 target gene sets of TFs associated with pluri- or multipotency. StemChecker presents an intuitive graphical display, as well as detailed statistical results in table format, which helps revealing transcriptionally regulatory programs, indicating the putative involvement of stemness-associated processes in diseases like cancer. Overall, StemChecker substantially expands the available repertoire of online tools, designed to assist the stem cell biology, developmental biology, regenerative medicine and human disease research community. StemChecker is freely accessible at http://stemchecker.sysbiolab.eu. Portuguese Fundacao para a Ciencia e Tecnologia (FCT) [RH/BPD/96890/2013, SFRH/BPD/70718/2010, PTDC/BIA/GEN/116519/2010, IF/00881/2013, PTDC/BIA-GEN/116519/2010]; Programa Doutoral ProRegeM-Mecanismos de Doenca e Medicina Regenerativa [PD/00117/2012] info:eu-repo/semantics/publishedVersion

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors (JUN), activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11)) as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.

Reinstating wild-type tumor suppressor p53 activity could be a valuable option for the treatment of cancer. To contribute to development of new treatment options for non-small cell lung cancer (NSCLC), we performed genome-wide siRNA screens for determinants of p53 activity in NSCLC cells. We identified many genes not previously known to be involved in regulating p53 activity. Silencing p53 pathway inhibitor genes was associated with loss of cell viability. The largest functional gene cluster influencing p53 activity was mRNA splicing. Prominent p53 activation was observed upon silencing of specific spliceosome components, rather than by general inhibition of the spliceosome. Ten genes were validated as inhibitors of p53 activity in multiple NSCLC cell lines: genes encoding the Ras pathway activator SOS1, the zinc finger protein TSHZ3, the mitochondrial membrane protein COX16, and the spliceosome components SNRPD3, SF3A3, SF3B1, SF3B6, XAB2, CWC22, and HNRNPL. Silencing these genes generally increased p53 levels, with distinct effects on CDKN1A expression, induction of cell cycle arrest and cell death. Silencing spliceosome components was associated with alternative splicing of MDM4 mRNA, which could contribute to activation of p53. In addition, silencing splice factors was particularly effective in killing NSCLC cells, albeit in a p53-independent manner. Interestingly, silencing SNRPD3 and SF3A3 exerted much stronger cytotoxicity to NSCLC cells than to lung fibroblasts, suggesting that these genes could represent useful therapeutic targets.

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo.

FlyRNAi (http://www.flyrnai.org), the database and website of the Drosophila RNAi Screening Center (DRSC) at Harvard Medical School, serves a dual role, tracking both production of reagents for RNA interference (RNAi) screening in Drosophila cells and RNAi screen results. The database and website is used as a platform for community availability of protocols, tools, and other resources useful to researchers planning, conducting, analyzing or interpreting the results of Drosophila RNAi screens. Based on our own experience and user feedback, we have made several changes. Specifically, we have restructured the database to accommodate new types of reagents; added information about new RNAi libraries and other reagents; updated the user interface and website; and added new tools of use to the Drosophila community and others. Overall, the result is a more useful, flexible and comprehensive website and database.

The discovery of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in a cell or an animal. The availability of genome-wide RNAi libraries targeting the mouse and human genomes has made it possible to carry out large scale, phenotype-based screens, which have yielded seminal information on diverse cellular processes ranging from virology to cancer biology. Today, several strategies are available to perform RNAi screens, each with their own technical and monetary considerations. Special care and budgeting must be taken into account during the design of these screens in order to obtain reliable results. In this review, we discuss a number of critical aspects to consider when planning an effective RNAi screening strategy, including selecting the right biological system, designing an appropriate selection scheme, optimizing technical aspects of the screen, and validating and verifying the hits. Similar to an artistic production, what happens behind the screen has a direct impact on its success.

The development of advanced functional genomic tools has paved the way for systematic investigations of biological processes in health and disease. In particular, the implementation of RNA interference (RNAi) as a genome-wide, loss-of-function screening tool has enabled scientists to probe the role for every gene in cellular assays and many new factors for various processes have been discovered employing RNAi screens in recent years. However, the results also demonstrate the complexity of biological systems and indicate that we are still a long way from understanding functional networks in depth. Nevertheless, RNAi screens present a powerful method to interrogate gene function in high-throughput and different methods to elicit RNAi in mammalian cells have been developed. Here, we describe steps that should be considered when planning an RNAi screen employing endoribonuclease prepared (e)siRNAs. We provide useful information on how to implement the screen and analyze the results. Furthermore, we discuss strategies for hit validation and present an outline on how to follow-up on verified hits to gain a molecular understanding of the underlying phenotypes.

Biological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations., United States. Dept. of Energy (Computational Science Graduate Fellowship), Leukemia & Lymphoma Society of America, Wellcome Trust (London, England) (Research Career Development Fellow), National Institutes of Health (U.S.) (Grant 1R01GM081871-01A1)

A major goal of aging research is to identify interventions that prolong lifespan in distantly related organisms. In recent years, genetic studies in both nematodes and rodents have reported that moderate inactivation of genes important for mitochondrial electron transport chain (ETC) function can promote longevity. We performed an RNAi screen to probe the role of ETC components in modulating lifespan in the fruit fly Drosophila melanogaster. In this Research Perspective, we discuss our findings and how they may relate to similar studies in worms and mice.

In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. Additionally, cancer cells exposed to these stresses are more resistant to therapies, more likely to metastasize and often are worse for patient prognosis. While the presence of these stresses is generally negative for cancer patients, since these stresses are mostly unique to the TME, they also offer an opportunity to develop more selective therapeutics. If we achieve a better understanding of the adaptive mechanisms cancer cells employ to survive the TME stresses, then hopefully we, as a scientific community, can devise more effective cancer therapeutics specifically targeting cancer cells under stress. To systematically identify genes that modulate cancer cell survival under stresses, we performed shRNA screens under hypoxia or lactic acidosis. From these screens, we discovered that genetic depletion of acetyl-CoA carboxylase alpha (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Furthermore, the loss of ACLY or ACC1 reduced the levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate was paradoxically increased under hypoxia when ACC1 or ACLY were depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulated the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

The role of Ki-67 in mitotic cell division has been a mystery. Extensive imaging reveals that this highly positively charged protein coats chromosomes to prevent them from coalescing. See Letter p.308 As chromosomes prepare for division by the process of mitosis, they undergo reorganization into separate bodies. Through an RNAi screen, Daniel Gerlich and colleagues demonstrate that this process is mediated by Ki-67 — a protein known as a cell proliferation marker and used in cancer diagnostics. After nuclear envelope disassembly, Ki-67 prevents chromosomes from collapsing into a single cluster by acting as a biological surfactant. In fact, many biophysical properties of this protein match those of surfactants, and it appears to be through these properties, rather than though a specific region of the protein, that Ki-67 ensures chromosome separation.

Stem cells are characterized by their potential for self-renewal and their capacity to differentiate into mature cells. These two key features emerge through the interplay of various factors within complex molecular networks. To provide researchers with a dedicated tool to investigate these networks, we have developed StemCellNet, a versatile web server for interactive network analysis and visualization. It rapidly generates focused networks based on a large collection of physical and regulatory interactions identified in human and murine stem cells. The StemCellNet web-interface has various easy-to-use tools for selection and prioritization of network components, as well as for integration of expression data provided by the user. As a unique feature, the networks generated can be screened against a compendium of stemness-associated genes. StemCellNet can also indicate novel candidate genes by evaluating their connectivity patterns. Finally, an optional dataset of generic interactions, which provides large coverage of the human and mouse proteome, extends the versatility of StemCellNet to other biomedical research areas in which stem cells play important roles, such as in degenerative diseases or cancer. The StemCellNet web server is freely accessible at http://stemcellnet.sysbiolab.eu. Portuguese Fundacao para a Ciencia e a Tecnologia [PTDC/BIA-GEN/116519/2010, SFRH/BPD/70718/2010, SFRH/BPD/96890/2013, PEst-OE/EQB/LA0023/2013] info:eu-repo/semantics/publishedVersion

Background MYC family members are among the most frequently deregulated oncogenes in human cancers, yet direct therapeutic targeting of MYC in cancer has been challenging thus far. Synthetic lethality provides an opportunity for therapeutic intervention of MYC-driven cancers. Methods A pooled kinase shRNA library screen was performed and next-generation deep sequencing efforts identified that PRKDC was synthetically lethal in cells overexpressing MYC. Genes and proteins of interest were knocked down or inhibited using RNAi technology and small molecule inhibitors, respectively. Quantitative RT-PCR using TaqMan probes examined mRNA expression levels and cell viability was assessed using CellTiter-Glo (Promega). Western blotting was performed to monitor different protein levels in the presence or absence of RNAi or compound treatment. Statistical significance of differences among data sets were determined using unpaired t test (Mann–Whitney test) or ANOVA. Results Inhibition of PRKDC using RNAi (RNA interference) or small molecular inhibitors preferentially killed MYC-overexpressing human lung fibroblasts. Moreover, inducible PRKDC knockdown decreased cell viability selectively in high MYC-expressing human small cell lung cancer cell lines. At the molecular level, we found that inhibition of PRKDC downregulated MYC mRNA and protein expression in multiple cancer cell lines. In addition, we confirmed that overexpression of MYC family proteins induced DNA double-strand breaks; our results also revealed that PRKDC inhibition in these cells led to an increase in DNA damage levels. Conclusions Our data suggest that the synthetic lethality between PRKDC and MYC may in part be due to PRKDC dependent modulation of MYC expression, as well as MYC-induced DNA damage where PRKDC plays a key role in DNA damage repair. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-944) contains supplementary material, which is available to authorized users.

Background Pancreatic cancer is a deadly disease with a five-year survival of less than 5%. A better understanding of the underlying biology may suggest novel therapeutic targets. Recent surveys of the pancreatic cancer genome have uncovered numerous new alterations; yet systematic functional characterization of candidate cancer genes has lagged behind. To address this challenge, here we have devised a highly-parallel RNA interference-based functional screen to evaluate many genomically-nominated candidate pancreatic cancer genes simultaneously. Results For 185 candidate pancreatic cancer genes, selected from recurrently altered genomic loci, we performed a pooled shRNA library screen of cell growth/viability across 10 different cell lines. Knockdown-associated effects on cell growth were assessed by enrichment or depletion of shRNA hairpins, by hybridization to barcode microarrays. A novel analytical approach (COrrelated Phenotypes for On-Target Effects; COPOTE) was used to discern probable on-target knockdown, based on identifying different shRNAs targeting the same gene and displaying concordant phenotypes across cell lines. Knockdown data were integrated with genomic architecture and gene-expression profiles, and selected findings validated using individual shRNAs and/or independent siRNAs. The pooled shRNA library design delivered reproducible data. In all, COPOTE analysis identified 52 probable on-target gene-knockdowns. Knockdown of known oncogenes (KRAS, MYC, SMURF1 and CCNE1) and a tumor suppressor (CDKN2A) showed the expected contrasting effects on cell growth. In addition, the screen corroborated purported roles of PLEKHG2 and MED29 as 19q13 amplicon drivers. Most notably, the analysis also revealed novel possible oncogenic functions of nucleoporin NUP153 (ostensibly by modulating TGFβ signaling) and Kruppel-like transcription factor KLF5 in pancreatic cancer. Conclusions By integrating physical and functional genomic data, we were able to simultaneously evaluate many candidate pancreatic cancer genes. Our findings uncover new facets of pancreatic cancer biology, with possible therapeutic implications. More broadly, our study provides a general strategy for the efficient characterization of candidate genes emerging from cancer genome studies.

Bunyaviruses are an emerging group of medically important viruses, many of which are transmitted from insects to mammals. To identify host factors that impact infection, we performed a genome-wide RNAi screen in Drosophila and identified 131 genes that impacted infection of the mosquito-transmitted bunyavirus Rift Valley fever virus (RVFV). Dcp2, the catalytic component of the mRNA decapping machinery, and two decapping activators, DDX6 and LSM7, were antiviral against disparate bunyaviruses in both insect cells and adult flies. Bunyaviruses 5′ cap their mRNAs by “cap-snatching” the 5′ ends of poorly defined host mRNAs. We found that RVFV cap-snatches the 5′ ends of Dcp2 targeted mRNAs, including cell cycle-related genes. Loss of Dcp2 allows increased viral transcription without impacting viral mRNA stability, while ectopic expression of Dcp2 impedes viral transcription. Furthermore, arresting cells in late S/early G2 led to increased Dcp2 mRNA targets and increased RVFV replication. Therefore, RVFV competes for the Dcp2-accessible mRNA pool, which is dynamically regulated and can present a bottleneck for viral replication.

Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens., Author Summary The mosquito immune system relies on innate humoral and cellular reactions to fight infections, including those by malaria parasites that must pass through mosquitoes before they can infect humans. Therefore, a detailed molecular understanding of these reactions could assist the design of new ways to control the spread of malaria and other mosquito-borne diseases. Here we use a technique to silence in mosquito cultured cells genes that are highly and/or specifically expressed in mosquito hemocytes, the equivalent of human white blood cells, as a means to investigate their function in reactions of the mosquito immune system. Our study identifies several novel regulators of immune reactions including phagocytosis, the engulfment and subsequent destruction of bacteria and other pathogens by hemocytes, the production of antimicrobial peptides, which directly kill or inhibit the proliferation of microbes, and the basal and induced production of an important complement regulator. Complement is a robust reaction of mosquitoes against malaria parasites and bacteria through phagocytosis, lysis or melanization (the enclosure of pathogens in a melanin capsule). We also reveal intriguing molecular connections between these reactions such as phagocytosis and regulation of complement. Our study provides novel insights into mosquito immune system and its reactions against infections.

Introduction: Despite optimal chemotherapy, radiotherapy and/or surgery, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. Therefore, there is a need for agents, such as radiosensitizers, that can improve the efficacy of current therapies. Using a whole-genome shRNA screen, we sought to identify and characterize novel gene products that may be involved in NSCLC radioresistance. We hypothesize that these proteins may represent novel targets for NSCLC radiosensitization. Materials and Methods: A whole-genome pooled retroviral shRNA screen was performed using the Hannon-Elledge library of 74,705 distinct shRNAs directed against over 18,000 genes, to identify gene knockdowns that showed cytotoxicity only in cells treated with fractionated radiation given daily Monday-Friday. To confirm targets identified in the whole-genome screen, we treated A549 and NCI-H460 NSCLC cells with siRNAs targeted against the genes of interest and assessed cell viability following irradiation using the CellTiter-Glo assay and clonogenic survival assays. Results: We identified six novel genes (BCAS1, c7orf24, CDC45L, KIAA0101, TCN1 and TNC) whose knockdown sensitized NSCLC cells to radiation. Of the six genes, siRNA knockdown of BCAS1 showed the most consistent increases in sensitivity to ionizing radiation. Analysis of cell viability following radiation revealed that BCAS1 knockdown resulted in a 50% additional decrease in cell viability in A549 and NCI-H460 cells following 8 and 4 Gy of ionizing radiation, respectively. We confirmed these results by clonogenic survival assays. To assess for tumor-specific expression in NSCLC, we compared expression data between matched normal tissue and NSCLC biopsy samples and found that BCAS1 is substantially more highly expressed in tumor tissues. Examination of affinity capture-mass spectroscopy data suggested that BCAS1 interacts with RUVBL2, a RuvB-like AAA ATPase, which has known functions in DNA damage and repair. Conclusions: BCAS1 emerged from a whole-genome RNAi screen as a potential target for improving the efficacy of radiotherapy in NSCLC. We demonstrated that BCAS1 is overexpressed in tumor tissues compared to normal lung, and that its knockdown consistently sensitizes NSCLC cell lines to radiation, validating the screen hit. Ongoing work suggests that BCAS1 may modulate DNA damage repair. Further studies to understand the mechanism by which BCAS1 is involved in radioresistance are necessary. Citation Format: Colin O’Leary, Peter Deraska, Alan D’Andrea, David Kozono. Whole-genome RNAi screen identified BCAS1 as a novel modifier of non-small cell lung carcinoma radioresistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1650.

Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegans strain CB55, which carries a truncating mutation in the unc-2 gene, the worm ortholog for the human CACNA1A. The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegans neurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/ CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway.

Background High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention. Results We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene. Conclusions RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.