mRNA localisation is a fundamental cellular process that provides a layer of protein regulation by directing protein synthesis in time and space. mRNAs may localise to cytosolic RNA granules, which contain distinct RNA and protein components. Cytosolic RNA granules are membrane-less regions that are often described as phase-separated from the surrounding cytosol, and appear to form via both specific and non-specific RNA-RNA, RNA-protein, and protein-protein interactions. RNA granules may regulate protein production by storing mRNAs in translationally repressed states, by encouraging either mRNA stabilisation or degradation, or by localising together specific mRNAs to produce proteins in the same multi-subunit complexes. Previous work in the Ashe lab has identified a novel set of RNA granules in yeast. These granules are distinct from other RNA granules; they do not co-localise with classical markers for other types of granules, they are present in unstressed yeast cells, and they contain mRNAs that are actively translated. Further work has demonstrated that there are at least three types of granules harbouring actively translated mRNAs, which contain different, specific mRNAs and display differing cytosolic RNA granule patterns. Experiments were therefore performed in this project involving the mutagenesis of select mRNAs to determine the localisation elements that drive this differential mRNA localisation. These experiments showed that the ORFs of two mRNAs were capable of driving the localisation pattern of their respective full-length mRNAs, suggesting that either the ORFs of both mRNAs contain RNA sequence and/or structural localisation elements, or that the production of the nascent protein is capable of driving the localisation of its own mRNA. To study mRNA localisation, microscopic visualisation techniques are used. Current systems all have potential drawbacks; the MS2 system involves modification of the mRNA structure, smFISH requires cell fixation/permeabilisation, and RNA aptamer systems require both mRNA modification and cell permeabilisation. A recently developed mRNA visualisation system, the dCas9-RNA targeting system, was therefore developed in this project for use in yeast. The dCas9 system is a live cell system that does not involve modification of the target mRNA, and is easily introduced into cells in a plasmid-based system. The dCas9 protein is specifically targeted to the mRNA of interest via a single-guide RNA, removing the need to modify the structure of the mRNA. The dCas9 system is shown here to be capable of accurately revealing patterns of mRNA localisation without affecting mRNA abundance, and is further used to target multiple mRNAs to identify novel localisation patterns. This dCas9 system has the potential to be used to rapidly screen endogenous, unmodified mRNA localisation patterns and study their dynamics in live cells.
|Date of Award||1 Aug 2021|
- The University of Manchester
|Supervisor||Mark Ashe (Supervisor) & Shane Herbert (Supervisor)|