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.
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.
BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.