Connecting Systemic RNAi to the Endomembrane System in Caenorhabditis elegans
Abstract: RNA interference (RNAi) is a gene regulation mechanism conserved among eukaryotes. To silence gene expression, RNAi relies on a short single-stranded guide RNA to steer the RNA-induced Silencing Complex (RISC) to mRNAs with guide strand-complementary sequences. RNAi is a highly membrane-associated process. The RISC complex is likely loaded at the rough Endoplasmic Reticulum, where it can bind to and degrade mRNAs. Components of the RISC complex also colocalize to late endosomes, and the efficiency of RNAi-mediated silencing is affected by changes in late endosome to lysosome fusion. RNAi can be systemic and inherited, effecting gene silencing in distal tissues and in the offspring.In this thesis, the model organism Caenorhabditis elegans was used to identify and characterize factors connecting systemic and inherited RNAi to the endomembrane system. We identify two SNARE proteins, SEC-22 and SYX-6, that both act as negative regulators of RNAi. SNAREs are necessary for vesicle fusion. Both SEC-22 and SYX-6 localize to late endosomes, and both interact with systemic RNAi protein SID-5 in a yeast two-hybrid (Y2H) screen. We find that in addition to its function in systemic RNAi, SID-5 is required for proper maturation of late endosomes. Furthermore, we identify the putative RNA-binding protein C12D8.1 as a novel regulator of RNAi inheritance. Mutant C12D8.1 animals will have enhanced inheritance of RNAi silencing, which negatively affects the ability of the progeny to silence new targets using RNAi. Finally, we describe a novel, object-based method for estimating significance in colocalization studies. This method helped us describe and quantify spatial relations between fluorophore-labeled proteins in situations where such analyses would otherwise be impossible.In conclusion, the work presented here further elucidates the connection between cellular RNAi, the endomembrane system, and the outside world.
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