Extracellular RNAs found in body fluids (serum, saliva, breast milk, etc.) are being investigated as sensitive sequence-specific markers for disease and as possible intercellular messengers. Understanding how specific RNAs are selected and exported and whether and how they are imported into target cells is integral to understanding their significance as biomarkers, as mediators of cellular functions, and as potential therapeutics. The discovery and characterization of RNA transporters that function to export and import double-stranded RNA (dsRNA) silencing signals in the model system C. elegans is providing molecular and genetic insights into this emerging field. In C. elegans, introduced dsRNA induces RNA interference (RNAi) that can spread between cells and tissues. Genetic analysis of systemic RNAi defective (Sid) mutants has identified not only RNA transporters, but regulatory and biogenesis factors that function in universal cell biological processes to support intercellular RNA transport. SID-1, a dsRNA-gated dsRNA-specific channel, confers the key dsRNA import and export activity to cells. Analysis of cultured cells expressing wild-type and mutant SID-1 proteins shows that nucleic acid transport by SID-1 is specific for dsRNA and sensitive to even minor chemical modifications to the dsRNA. Similar assays indicate that fish, mouse, and human SID-1 homologs also transport dsRNA. If the human homologs are equally specific for dsRNA and intolerant to modification, then the myriad of RNA modifications designed to stabilize therapeutic silencing RNAs in sera and reduce their immunogenicity may contribute to the difficulty of delivering these RNAs into cells. In AIM 1 we will determine the effect of common natural and unnatural RNA modifications on dsRNA transport in both C. elegans and cultured cells. These results will be directly applicable to the design of effective human therapeutic siRNAs. Mammalian extracellular RNAs are often found within exosomes, secreted vesicles derived from late endosome/multivesicular bodies (LE/MVB). SID-5 is a novel LE/MVB associated protein required for efficient export of RNA silencing signals, indicating that exosomes may transport RNA silencing signals in C. elegans. In AIM 2 we propose to characterize the activity, localization, and function of new SID proteins, including SID-1 and SID-5 interacting proteins to gain insight into the biological processes important for RNA export. These results will provide a genetic foundation and mechanistic understanding for the emerging fields of exosome biology and extracellular RNAs. In AIM 3 we propose to exploit the experimental expertise and resources developed in the Hunter lab to directly isolate endogenous extracellular RNAs and then to use the well established molecular genetic tools available in C. elegans to characterize their expression, transport, and function. Understanding how extracellular RNA signals contribute to animal physiology is key to understanding their significance as biomarkers and therapeutic targets for cancer therapy and other biological processes. !