An RNAi screen in Drosophila cells for the identification of regulators of Stress Granules? dynamics

MARCELO PEREZ PEPE; LORENA B BENSEÑOR; NATALIA CONTRERAS; MARIELA LOSCHI; GRACIELA L BOCCACCIO

Oulu

Congreso; OXYGEN 2013 Meeting; 2013

Stress granules (SGs) are cytoplasmic foci that form transiently during hipoxia and other forms of cellular stress. SGs contain repressed mRNAs and RNA-binding proteins involved in reprogramming mRNA translation and decay, and also sequester key modulators of cell survival and proliferation, all this helping to avoid apoptosis. SGs are related to another kind of ubiquitous mRNA silencing foci termed Processing Bodies (PBs) and to abnormal protein aggregates present in neurodegenerative conditions (Baez et al., J Cell Biol 2011; Martinez Tosar et al., FIB 2012; Pascual et al., CIB 2012). SG assembly and disassembly is a multi-step process that depends on: i) translation inhibition; ii) retrograde transport mediated by dynein; iii) aggregation by specific protein domains and iv) dispersion mediated by kinesin and specific chaperones (Thomas et al, MBoC 2005; JCS 2009; Loschi et al., JCS 2009; Thomas et al., Cell Signal 2011). We performed a high-throughput RNAi screen in Drosophila cells to identify the signaling pathways regulators of SG dynamics. To analyze the numerous confocal images, we developed a MATLAB script (Perez-Pepe et al., PLoS 2012) that allowed the identification of 32 positive regulators and 15 negative regulators of SG formation. As expected, stress-activated protein kinases and eIF2alpha kinases mediate SG assembly, whereas the phosphatase PP1α affects SG dissolution. Ten novel genes facilitate SG assembly and 7 genes encoding unknown functions or related to neurodegeneration affect SG disassembly. This kinase-phosphatase screen opens new lines of research on SG dynamics.