Signaling pathways in Drosophila immunity

University dissertation from Stockholm : Department of Molecular Biology and Functional Genomics, Stockholm University

Abstract: Drosophila relies on innate immunity to protect itself from its hostile environment throughout its life cycle. Despite the remarkable progress in understanding many aspects of Drosophila immunity, there are still big gaps in our knowledge. The general aim of this thesis was to gain a better understanding about the regulatory mechanisms controlling gene expression in Drosophila, with a focus on immunity. To enable isolation of Drosophila genes involved in immunity, we developed a method that allows visualization of immune gene expression in large number of embryos.  Reporter gene expression in wild type and mutant embryos was used to validate this approach, which should be a valuable complement to existing genetic and RNAi screens. Cactus, the Drosophila IκB protein, is known as a cytoplasmic inhibitor of Dif and Dorsal. We discovered that Cactus is also present in the cell nucleus. In response to Toll pathways signaling, cytoplasmic Cactus degrades rapidly in a proteasome-dependent manner, while a nuclear form of Cactus is stable and persists throughout signaling. This suggests that Cactus also has a function in the nucleus.A genome-wide RNAi-based screen was performed in cultured S2 cells. Several novel components of NF-κB pathways were isolated as putative regulators of Drosophila immunity. One of them, the G protein-coupled receptor kinase-2 (Gprk2), was shown to be required for Drosomycin expression and for resistance to infection. Gprk2 interacts with Cactus, but is not required for Cactus degradation upon Toll pathway activation.  The dpld/wech gene was previously found to affect periferal nervous system development. Here, we show that wech belongs to the LIN-41 subclade of the TRIM protein superfamily, and contains target sites for microRNAs. Genetic and cell transfection assays confirmed that wech expression is regulated by the microRNA let-7. This seems to be a conserved regulatory mechanism throughout the LIN-41 subclade. 

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