Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-beta. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-beta and BMP into the nucleus.
Although classical genetic and biochemical approaches have identified hundreds of proteins that function in the dynamic remodeling of cell shape in response to upstream signals, there is currently little systems-level understanding of the organization and composition of signaling networks that regulate cell morphology. We have developed quantitative morphological profiling methods to systematically investigate the role of individual genes in the regulation of cell morphology in a fast, robust, and cost-efficient manner. We analyzed a compendium of quantitative morphological signatures and described the existence of local signaling networks that act to regulate cell protrusion, adhesion, and tension.
Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.
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.
Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.
Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.
Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs or downstream pathway components such as Ras and Raf. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components-in multiple cell types with different RTK ligands and oncogenic stimuli-validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.
Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.
Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.
RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.
Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.
Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.
The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.
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.
The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.
Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.
The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.
This chapter describes the method used to conduct high-throughput screening (HTs) by RNA interference in Drosophila tissue culture cells. It covers four main topics: (1) a brief description of the existing platforms to conduct RNAi-screens in cell-based assays; (2) a table of the Drosophila cell lines available for these screens and a brief mention of the need to establish other cell lines as well as cultures of primary cells; (3) a discussion of the considerations and protocols involved in establishing assays suitable for HTS in a 384-well format; and (A) a summary of the various ways of handling raw data from an ongoing screen, with special emphasis on how to apply normalization for experimental variation and statistical filters to sort out noise from signals.
A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.
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.