Jennifer A Philips, Eric J Rubin, and Norbert Perrimon. 2005. “
Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection.” Science, 309, 5738, Pp. 1251-3.
AbstractCertain 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.
2005_Science_Philips.pdf Ramanuj DasGupta, Ajamete Kaykas, Randall T Moon, and Norbert Perrimon. 2005. “
Functional genomic analysis of the Wnt-wingless signaling pathway.” Science, 308, 5723, Pp. 826-33.
AbstractThe 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.
2005_Science_DasGupta.pdf Kent Nybakken, Steven A Vokes, Ting-Yi Lin, Andrew P McMahon, and Norbert Perrimon. 2005. “
A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway.” Nat Genet, 37, 12, Pp. 1323-32.
AbstractMembers 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.
2005_Nat Gene_Nybakken.pdf Supplemental Files.zip Gyeong-Hun Baeg, Rui Zhou, and Norbert Perrimon. 2005. “
Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila.” Genes Dev, 19, 16, Pp. 1861-70.
AbstractThe 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.
2005_Genes Dev_Baeg.pdf Supplemental Files.zip Addendum.pdf Hervé Agaisse, Laura S Burrack, Jennifer A Philips, Eric J Rubin, Norbert Perrimon, and Darren E Higgins. 2005. “
Genome-wide RNAi screen for host factors required for intracellular bacterial infection.” Science, 309, 5738, Pp. 1248-51.
AbstractMost 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.
2005_Science_Agaisse.pdf Sara Cherry, Tammy Doukas, Susan Armknecht, Sean Whelan, Hui Wang, Peter Sarnow, and Norbert Perrimon. 2005. “
Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.” Genes Dev, 19, 4, Pp. 445-52.
AbstractThe 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.
2005_Genes Dev_Cherry.pdf Supplement.pdf Susan Armknecht, Michael Boutros, Amy Kiger, Kent Nybakken, Bernard Mathey-Prevot, and Norbert Perrimon. 2005. “
High-throughput RNA interference screens in Drosophila tissue culture cells.” Methods Enzymol, 392, Pp. 55-73.
AbstractThis 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.
2005_Methods Enzym_Armknecht.pdf