Investigating the effects of oxidative stress on extracellular vesicle function and RNA cargo loading

Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed structures which have been suggested to play a critical role in maintaining intracellular homeostasis through the elimination of unnecessary biological material from EV-producing cells, and as a delivery system enabling intercellular communication between distant cells. Although extensive study has revealed many RNA biotypes that are associated with EVs, and has begun to elucidate the mechanisms of their packaging and delivery, little is currently known about how these processes are conserved in different species and under different cellular conditions. In this thesis, an in vitro Drosophila model in S2R+ cells was set up to investigate EV biology under normal and stress conditions, as a complement to an in vivo system being set up in Drosophila larvae. Under hydrogen peroxide-induced oxidative stress, an increase to EV number was seen, whilst no changes to size or protein marker composition were evident. Preliminary work indicated that these stress-cell- derived EVs acted to induce cell death in recipient cells. Characterisation of the total RNA content of cells and EV samples under control and oxidative stress conditions revealed that EV RNA was not representative of parental cells, but was instead enriched for ribosomal RNA, Copia retrotransposon, snoRNA, and RNAs relating to cytoskeletal and developmental functions. Investigation of potential loading motifs in mRNA 3’UTRs found no sequences which could differentiate mRNA packaging under stress conditions. Further exploration of mRNA loading in EVs found no preference for short mRNA transcripts, but an enrichment of intronic and untranslated regions, and 3’ end degradation, under stress. A striking increase in base mismatch in the RNA of EV preparations was seen under stress, possibly indicative of targeting of aberrant RNA to EVs, or of RNA modification. These results present strong evidence that the RNA content of Drosophila EVs is altered under oxidative stress and provides several avenues for further investigation of mechanisms by which it may occur. Furthermore, these data indicate the potential increased importance that EVs may play in metabolic and signalling functions under different cellular stress conditions, which has great relevance for disease pathology.