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The shRNAs were grouped in nine sublibraries of 55,000 shRNAs each, based on annotated biological functions (Extended Experimental Procedures). For our first application of the genome-wide screening approach, we also used ricin, as it should give access to the rich biology of host pathways exploited by this toxin (Lord et?al., 2005; Sandvig et?al., 2010; Spooner and Lord, 2012). Specifically, ricin is internalized by endocytosis and traffics retrogradely through the secretory pathway to the ER, where its A and B subunits are dissociated. The catalytic A subunit is then retrotranslocated click here to the cytoplasm, where it depurinates a single base in the 28S rRNA, shutting down translation and leading to apoptosis (Figure?2A). We defined a set of hit genes based on false discovery rate (FDR; Storey and Tibshirani, 2003); this set contained the 73 strongest protective hits (FDR NAD hits included genes either acting in the secretory pathway or otherwise expected based on known ricin biology. In addition, we tagged several poorly characterized hit genes with GFP, expressed them from their native chromosomal context in BACs (Poser et?al., 2008), and confirmed that they were localized to secretory pathway organelles (Figure?S3). We found learn more that many of the top hits in the screen are also known to exist in physical complexes with each other, with strong protection upon knockdown of components of COPII, TRAPP, and GARP and strong sensitization upon knockdown of components of COPI, the ribosome, and the proteasome. Taken together, the above results illustrate the specificity and robustness of the hits identified by our approach. Consistent with results from previous ricin screens and individual gene studies, we found that the early endocytic factors clathrin and Rab5 (Moreau et?al., 2011) were required for ricin toxicity, as well as STX16, a snare protein involved in vesicle fusion at the TGN (Amessou et?al., 2007). Among the most strongly enriched were components of the GARP complex known to be required for tethering endosome-derived vesicles to the Golgi (Bonifacino and Hierro, 2011). Knockdowns of several (but not all, see below) components of the vesicle-tethering TRAPP complex were among the most strongly protective. Surprisingly, a large number of components of the COPII machinery required for anterograde vesicle budding from the ER were strongly protective against ricin when knocked down, which has not been previously observed. It is likely that shutdown of ER-Golgi trafficking (and consequent Golgi collapse) prevents ER delivery of ricin.