Identification of mRNA features which facilitate their long-range transfer in an in vivo Drosophila model

Extracellular vesicles (EV) are membrane bound carriers released by cells to mediate important aspects of intercellular communication; however, the basic principles of EV-biogenesis, in particular the mechanisms regulating mRNA EV-loading, are poorly characterised. Furthermore, a limited repertoire of tools has thus far made these processes challenging to research. To address these fundamental biological questions, an RNA-sequencing analysis of Drosophila EVs and the cells from which they derive was carried out. A bioinformatic pipeline was subsequently developed with the aim of identifying molecular features within the 3'UTR of mRNAs which may encourage their EV incorporation and long-range transfer. In addition, the role of these EV-enriched features was assessed in vivo, in complex Drosophila tissues, using an adapted version of the Cre-LoxP reporter to visualise long-range cell communication. These analyses revealed a distinct mRNA profile for Drosophila cells versus EVs and identified unique EV-enriched 3'UTR motifs. Motifs were preferentially arranged in distinct combinations and tended to occur in specific RNA secondary structures. These features may modulate their interaction with trans-acting factors, such as RNA binding proteins, with sequence similarity observed between these novel motifs and well-established RNA binding-protein binding sites. Subsequent in vivo analyses highlighted the potential for mRNA 3'UTR modifications to successfully modulate long-range mRNA transfer in complex tissues. EV-enriched motifs within these 3'UTR modifications, however, may only play a contributing role. Future work is needed to assess the effect of specific 3'UTR modifications on mRNA stability, length and GC content, as these factors have the potential to influence long-range mRNA transfer. These investigations provide novel insights into the biological properties facilitating mRNA transfer between Drosophila cells without physical contact. Importantly, these biological features may be evolutionarily conserved, given that similar mRNA properties have been described in mammalian cells. Therefore, these findings could establish the basis to efficiently modulate mRNA transfer and cell function at a distance.