Norbert Perrimon and Bernard Mathey-Prevot. 2007. “
Applications of high-throughput RNA interference screens to problems in cell and developmental biology.” Genetics, 175, 1, Pp. 7-16.
AbstractRNA interference (RNAi) in tissue culture cells has emerged as an excellent methodology for identifying gene functions systematically and in an unbiased manner. Here, we describe how RNAi high-throughput screening (HTS) in Drosophila cells are currently being performed and emphasize the strengths and weaknesses of the approach. Further, to demonstrate the versatility of the technology, we provide examples of the various applications of the method to problems in signal transduction and cell and developmental biology. Finally, we discuss emerging technological advances that will extend RNAi-based screening methods.
2007_Genetics_Perrimon.pdf Yousang Gwack, Sonal Srikanth, Stefan Feske, Fernando Cruz-Guilloty, Masatsugu Oh-hora, Daniel S Neems, Patrick G Hogan, and Anjana Rao. 2007. “
Biochemical and functional characterization of Orai proteins.” J Biol Chem, 282, 22, Pp. 16232-43.
AbstractStimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.
2007_J Bio Chem_Gwack.pdf Supplement.pdf Ramanuj DasGupta, Kent Nybakken, Matthew Booker, Bernard Mathey-Prevot, Foster Gonsalves, Binita Changkakoty, and Norbert Perrimon. 2007. “
A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila.” Genome Biol, 8, 9, Pp. R203.
AbstractOff-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.
Nadire Ramadan, Ian Flockhart, Matthew Booker, Norbert Perrimon, and Bernard Mathey-Prevot. 2007. “
Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.” Nat Protoc, 2, 9, Pp. 2245-64.
AbstractThis 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.
2007_Nat Prot_Ramadan.pdf Jianrong Lu, Marie-Laure Ruhf, Norbert Perrimon, and Philip Leder. 2007. “
A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression.” Proc Natl Acad Sci U S A, 104, 22, Pp. 9381-6.
AbstractRegulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, we have conducted a genome-wide RNAi screen in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related CG16975/dSfmbt. These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth.
2007_PNAS_Lu.pdf Supplement.pdf Eric J Wagner, Brandon D Burch, Ashley C Godfrey, Harmony R Salzler, Robert J Duronio, and William F Marzluff. 2007. “
A genome-wide RNA interference screen reveals that variant histones are necessary for replication-dependent histone pre-mRNA processing.” Mol Cell, 28, 4, Pp. 692-9.
AbstractMetazoan replication-dependent histone mRNAs are not polyadenylated and instead end in a conserved stem loop that is the cis element responsible for coordinate posttranscriptional regulation of these mRNAs. Using biochemical approaches, only a limited number of factors required for cleavage of histone pre-mRNA have been identified. We therefore performed a genome-wide RNA interference screen in Drosophila cells using a GFP reporter that is expressed only when histone pre-mRNA processing is disrupted. Four of the 24 genes identified encode proteins also necessary for cleavage/polyadenylation, indicating mechanistic conservation in formation of different mRNA 3' ends. We also unexpectedly identified the histone variants H2Av and H3.3A/B. In H2Av mutant cells, U7 snRNP remains active but fails to accumulate at the histone locus, suggesting there is a regulatory pathway that coordinates the production of variant and canonical histones that acts via localization of essential histone pre-mRNA processing factors.
2007_Mol Cell_Wagner.pdf Supplement.pdf Caroline H Yi, Dodzie K Sogah, Michael Boyce, Alexei Degterev, Dana E Christofferson, and Junying Yuan. 2007. “
A genome-wide RNAi screen reveals multiple regulators of caspase activation.” J Cell Biol, 179, 4, Pp. 619-26.
AbstractApoptosis 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.
2007_J Cell Bio_Yi.pdf Supplemental Files.zip Lan Xu, Xiaohao Yao, Xiaochu Chen, Peiyuan Lu, Biliang Zhang, and Tony Y Ip. 2007. “
Msk is required for nuclear import of TGF-{beta}/BMP-activated Smads.” J Cell Biol, 178, 6, Pp. 981-94.
AbstractNuclear 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.
2007_J Cell Bio_Xu.pdf Supplement.pdf Chris Bakal, John Aach, George Church, and Norbert Perrimon. 2007. “
Quantitative morphological signatures define local signaling networks regulating cell morphology.” Science, 316, 5832, Pp. 1753-6.
AbstractAlthough 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.
2007_Science_Bakal.pdf Isabelle Derré, Marc Pypaert, Alice Dautry-Varsat, and Hervé Agaisse. 2007. “
RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection.” PLoS Pathog, 3, 10, Pp. 1446-58.
AbstractChlamydia 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.
2007_PLOS Path_Derre.pdf Supplemental Files.zip