Investigating RNA localisation to translation factories in Saccharomyces cerevisiae

Abstract

The targeting of specific mRNAs to particular subcellular locations is a widespread post-transcriptional mechanism regulating gene expression. Typically, mRNA localisation is coupled with tight translational control. For example, translationally repressed mRNAs can be localised to specific granules such as stress granules and P-bodies, providing mRNA storage during stress. Recently, the Ashe lab have used an MS2/ GFP tethering system in yeast called m-Tag to follow specific mRNAs and their localisation to previously undiscovered granules. In contrast to the aforementioned granules, these foci contain actively translated, rather than translationally repressed mRNAs. Moreover, mRNAs encoding related proteins often co-localise to the same site or translation factory: many glycolytic mRNAs co-localise to the same foci, whilst mRNAs encoding components of the translation machinery co-localise to another, distinct locus. As such, it is possible that these granules play roles in highly efficient yet coordinated translation, allowing cells to harmonize the production of components from the same protein complex or metabolic pathway. In this thesis a single-molecule microscopy technique, smFISH, has been used to further interrogate these novel mRNP granules, providing a high-resolution view of these sites in wild-type cells. These experiments confirmed the localisation of multiple endogenous unmodified mRNAs to foci in actively growing cells, validating data from the m-Tag approach. In addition, this approach provided novel insights relating to these translation factories. It uncovered their mRNA stoichiometry and identified that these granules harbour significant proportions of the cellular pool of the localising RNAs. In addition, smFISH studies have revealed that translation factories promote co-localisation between mRNAs encoding related proteins. Although the specific mechanism by which mRNAs localise to these sites is not yet known, analysis of hits identified from high-throughput approaches including a synthetic genetic array and a proteomic screen have offered significant insight into the mechanisms that underpin translation factory formation/maintenance. These experiments reveal that mRNA localisation to these sites is reliant on active translation and co-translational nascent protein folding and that the factories are acutely sensitive to perturbations in global protein folding within the cell. In sum, this work highlights that mRNA translation might be compartmentalised into phase-separated biological condensates and that such an arrangement could assist in protein complex formation and co-ordinated translational regulation.

Date of Award	31 Dec 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Simon Hubbard (Supervisor) & Mark Ashe (Supervisor)