Nearly 1.7 billion people are infected with Mycobacterium tuberculosis. Its ability to survive intracellularly is thought to be central to its success as a pathogen, but how it does this is poorly understood. Using a Drosophila model of infection, we identify three host cell activities, Rab7, CG8743, and the ESCRT machinery, that modulate the mycobacterial phagosome. In the absence of these factors the cell no longer restricts growth of the non-pathogen Mycobacterium smegmatis. Hence, we identify factors that represent unique vulnerabilities of the host cell, because manipulation of any one of them alone is sufficient to allow a nonpathogenic mycobacterial species to proliferate. Furthermore, we demonstrate that, in mammalian cells, the ESCRT machinery plays a conserved role in restricting bacterial growth.
Mitochondria are integral components of cellular calcium (Ca2+) signaling. Calcium stimulates mitochondrial adenosine 5'-triphosphate production, but can also initiate apoptosis. In turn, cytoplasmic Ca2+ concentrations are regulated by mitochondria. Although several transporter and ion-channel mechanisms have been measured in mitochondria, the molecules that govern Ca2+ movement across the inner mitochondrial membrane are unknown. We searched for genes that regulate mitochondrial Ca2+ and H+ concentrations using a genome-wide Drosophila RNA interference (RNAi) screen. The mammalian homolog of one Drosophila gene identified in the screen, Letm1, was found to specifically mediate coupled Ca2+/H+ exchange. RNAi knockdown, overexpression, and liposome reconstitution of the purified Letm1 protein demonstrate that Letm1 is a mitochondrial Ca2+/H+ antiporter.
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.
A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.
Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. Here we report the results of a genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI) complex, the ribosome and the proteasome. We identified the multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, as Hh regulators. We also identified a large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, we showed that several of these genes have conserved roles in mammalian Hh signaling.
This protocol describes the various steps and considerations involved in planning and carrying out RNA interference (RNAi) genome-wide screens in cultured Drosophila cells. We focus largely on the procedures that have been modified as a result of our experience over the past 3 years and of our better understanding of the underlying technology. Specifically, our protocol offers a set of suggestions and considerations for screen optimization and a step-by-step description of the procedures successfully used at the Drosophila RNAi Screening Center for screen implementation, data collection and analysis to identify potential hits. In addition, this protocol briefly covers postscreen analysis approaches that are often needed to finalize the hit list. Depending on the scope of the screen and subsequent analysis and validation involved, the full protocol can take anywhere from 3 months to 2 years to complete.
The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA, and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes.
Akt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)-dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K-independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt-TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo.
Reactive Oxygen Species (ROS) are a natural by-product of cellular growth and proliferation, and are required for fundamental processes such as protein-folding and signal transduction. However, ROS accumulation, and the onset of oxidative stress, can negatively impact cellular and genomic integrity. Signalling networks have evolved to respond to oxidative stress by engaging diverse enzymatic and non-enzymatic antioxidant mechanisms to restore redox homeostasis. The architecture of oxidative stress response networks during periods of normal growth, and how increased ROS levels dynamically reconfigure these networks are largely unknown. In order to gain insight into the structure of signalling networks that promote redox homeostasis we first performed genome-scale RNAi screens to identify novel suppressors of superoxide accumulation. We then infer relationships between redox regulators by hierarchical clustering of phenotypic signatures describing how gene inhibition affects superoxide levels, cellular viability, and morphology across different genetic backgrounds. Genes that cluster together are likely to act in the same signalling pathway/complex and thus make "functional interactions". Moreover we also calculate differential phenotypic signatures describing the difference in cellular phenotypes following RNAi between untreated cells and cells submitted to oxidative stress. Using both phenotypic signatures and differential signatures we construct a network model of functional interactions that occur between components of the redox homeostasis network, and how such interactions become rewired in the presence of oxidative stress. This network model predicts a functional interaction between the transcription factor Jun and the IRE1 kinase, which we validate in an orthogonal assay. We thus demonstrate the ability of systems-biology approaches to identify novel signalling events.
Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.
Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.
During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.
To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.
Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts.
Although a large number of actin-binding proteins and their regulators have been identified through classical approaches, gaps in our knowledge remain. Here, we used genome-wide RNA interference as a systematic method to define metazoan actin regulators based on visual phenotype. Using comparative screens in cultured Drosophila and human cells, we generated phenotypic profiles for annotated actin regulators together with proteins bearing predicted actin-binding domains. These phenotypic clusters for the known metazoan "actinome" were used to identify putative new core actin regulators, together with a number of genes with conserved but poorly studied roles in the regulation of the actin cytoskeleton, several of which we studied in detail. This work suggests that although our search for new components of the core actin machinery is nearing saturation, regulation at the level of nuclear actin export, RNA splicing, ubiquitination, and other upstream processes remains an important but unexplored frontier of actin biology.