A genome-wide RNAi screen reveals multiple regulators of caspase activation

Apoptosis is an evolutionally conserved cellular suicide mechanism that can be activated in response to a variety of stressful stimuli. Increasing evidence suggests that apoptotic regulation relies on specialized cell death signaling pathways and also integrates diverse signals from additional regulatory circuits, including those of cellular homeostasis. We present a genome-wide RNA interference screen to systematically identify regulators of apoptosis induced by DNA damage in Drosophila melanogaster cells. We identify 47 double- stranded RNA that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. Our data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death.     

A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila

Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.     

A screen for modifiers of hedgehog signaling in Drosophila melanogaster identifies swm and mts

Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. We performed an enhancer/suppressor screen in Drosophila to identify novel components of the pathway and identified 26 autosomal regions that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway-patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. We show that mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn(+) finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity.     

A genome-wide RNAi screen for genes important for proliferation of cultured Drosophila cells at low temperature identifies the Ball/VRK protein kinase

A change in ambient temperature is predicted to disrupt cellular homeostasis by affecting all cellular processes in an albeit non-uniform manner. Diffusion is generally less temperature-sensitive than enzymes, for example, and each enzyme has a characteristic individual temperature profile. The actual effects of temperature variation on cells are still poorly understood at the molecular level. Towards an improved understanding, we have performed a genome-wide RNA interference screen with S2R?+?cells. This Drosophila cell line proliferates over a temperature range comparable to that tolerated by the parental ectothermic organism. Based on effects on cell counts and cell cycle profile after knockdown at 27 and 17 °C, respectively, genes were identified with an apparent greater physiological significance at one or the other temperature. While 27 °C is close to the temperature optimum, the substantially lower 17 °C was chosen to identify genes important at low temperatures, which have received less attention compared to the heat shock response. Among a substantial number of screen hits, we validated a set successfully in cell culture and selected ballchen for further evaluation in the organism. This gene encodes the conserved metazoan VRK protein kinase that is crucial for the release of chromosomes from the nuclear envelope during mitosis. Our analyses in early embryos and larval wing imaginal discs confirmed a higher requirement for ballchen function at temperatures below the optimum. Overall, our experiments validate the genome-wide screen as a basis for future characterizations of genes with increased physiological significance at the lower end of the readily tolerated temperature range.

     

A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion

Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export.     

Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance

Evasion from apoptosis is a hallmark of cancer, and recent success using targeted therapeutics underscores the importance of identifying anti-apoptotic survival pathways. Here we utilize RNA interference (RNAi) to systematically screen the kinase and phosphatase component of the human genome. In addition to known kinases, we identified several new survival kinases. Interestingly, numerous phosphatases and associated regulatory subunits contribute to cell survival, revealing a previously unrecognized general role for phosphatases as negative regulators of apoptosis. We also identified a subset of phosphatases with tumour-suppressor-like activity. Finally, RNAi targeting of specific protein kinases sensitizes resistant cells to chemotherapeutic agents. The development of inhibitors that target these kinases or phosphatases may lead to new anti-cancer strategies.     

A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.     

Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand

The Hedgehog (Hh) signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp) and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand.     

In vivo RNAi screen identifies candidate signaling genes required for collective cell migration in Drosophila ovary

Collective migration of loosely or closely associated cell groups is prevalent in animal development, physiological events, and cancer metastasis. However, our understanding of the mechanisms of collective cell migration is incomplete. Drosophila border cells provide a powerful in vivo genetic model to study collective migration and identify essential genes for this process. Using border cell-specific RNAi-silencing in Drosophila, we knocked down 360 conserved signaling transduction genes in adult flies to identify essential pathways and genes for border cell migration. We uncovered a plethora of signaling genes, a large proportion of which had not been reported for border cells, including Rack1(Receptor of activated C kinase) and brk(brinker), mad(mother against dpp), and sax(saxophone), which encode three components of TGF-β signaling. The RNAi knock down phenotype was validated by clonal analysis of Rack1 mutants. Our data suggest that inhibition of Src activity by Rack1 may be important for border cell migration and cluster cohesion maintenance. Lastly, results from our screen not only would shed light on signaling pathways involved in collective migration during embryogenesis and organogenesis in general, but also could help our understanding for the functions of conserved human genes involved in cancer metastasis.     

An RNAi screen identifies genes that regulate GABA synapses

GABA synapses play a critical role in many aspects of circuit development and function. For example, conditions that perturb GABA transmission have been implicated in epilepsy. To identify genes that regulate GABA transmission, we performed an RNAi screen for genes whose inactivation increases the activity of C. elegans body muscles, which receive direct input from GABAergic motor neurons. We identified 90 genes, 21 of which were previously implicated in seizure syndromes, suggesting that this screen has effectively identified candidate genes for epilepsy. Electrophysiological recordings and imaging of excitatory and inhibitory synapses indicate that several genes alter muscle activity by selectively regulating GABA transmission. In particular, we identify two humoral pathways and several protein kinases that modulate GABA transmission but have little effect on excitatory transmission at cholinergic neuromuscular junctions. Our data suggest these conserved genes are components of signaling pathways that regulate GABA transmission and consequently may play a role in epilepsy and other cognitive or psychiatric disorders.     

Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation

Unlike mammalian Toll-like Receptors, the Drosophila Toll receptor does not interact directly with microbial determinants but is rather activated upon binding a cleaved form of the cytokine-like molecule Spatzle (Spz). During the immune response, Spz is thought to be processed by secreted serine proteases (SPs) present in the hemolymph that are activated by the recognition of gram-positive bacteria or fungi . In the present study, we have used an in vivo RNAi strategy to inactivate 75 distinct Drosophila SP genes. We then screened this collection for SPs regulating the activation of the Toll pathway by gram-positive bacteria. Here, we report the identification of five novel SPs that function in an extracellular pathway linking the recognition proteins GNBP1 and PGRP-SA to Spz. Interestingly, four of these genes are also required for Toll activation by fungi, while one is specifically associated with signaling in response to gram-positive bacterial infections. These results demonstrate the existence of a common cascade of SPs upstream of Spz, integrating signals sent by various secreted recognition molecules via more specialized SPs.     

A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.