Abstract
Exosomes are small vesicles containing proteins, miRNAs and mRNAs. Exosomes are secreted and taken up by neighbouring cells and the contents move into the recipient cell, where they are biologically active (e.g. the mRNA can be translated). Exosome contents are not present at the same proportions of a cell, meaning there must be active sorting of contents into exosomes. Currently the signals that determine which mRNAs are localised into exosomes are of great interest, as being able to manually add a localisation signal would allow for movement of contents that otherwise could not be spread between cells (such as the mRNA of a transmembrane protein). One such signal has been identified in glioblastoma mRNA 3' UTRs and the results replicated in HEK293 cells, however, is yet to be tested in other cell types.
Astrocytes are a cell type that perform regulatory functions in the brain, by providing nutrients and aiding the development of neurons. They able to actively divide, unlike neurons meaning they are much easier to grow in a cell culture environment for study and research.
This project's aim was to study mRNA isolated from astrocyte-derived exosomes, develop bioinformatic methods to determine any loading signals present in exosome mRNAs using external datasets, identify these signals present in astrocyte exosomes and then validate these signals in vitro. Protocols for enriching a mixed neural cell culture for astrocytes were successfully established and exosomes were isolated. Unfortunately the RNA extractions from exosomes were unsuccessful. The in silico analysis involved the processing of 8 individual datasets from independent labs, re-analysing all raw data and generating new datasets containing mRNA 3' UTRs to suit this projects aims. RNA sequence motifs, miRNA binding sites, and RNA secondary structures were all carefully analysed, revealing large differences in putative 3' UTR elements present in exosome mRNAs between different cell types. These data suggest that mRNA localisation to exosomes may be both species and cell type specific.