The role of mRNA localisation and oxidative stress in prion formation in yeast

Abstract

Prions are one of many forms of misfolded protein implicated in disease. Prions are heritable, misfolded versions of wild-type proteins which can induce further mis-folding of their wild-type counterparts. Prion diseases share many similarities with other neurodegenerative diseases which can, in many cases, be considered diseases of aging. Multiple prion proteins are well characterised in yeast including Sup35p, which forms [PSI+] and Rnq1p forming [PIN+] and yeast is thus an ideal model for studying protein misfolding and aggregation. While much is known about prion transmission, less is understood about the initial misfolding event which gives rise to the heritable prion form. Previous work has shown that oxidative stress can increase prion formation frequency in yeast, however the mechanism by which this occurs is not yet understood. The overall aim of the work described in this thesis was to determine whether the localisation of prion encoding mRNAs influences the probability of protein misfolding and prion formation and whether this is affected by oxidative stress. Using MS2-tagging and live cell imaging, RNQ1 and SUP35 mRNAs were found to localise to foci under normal and oxidative stress conditions. Foci localisation was further increased in response to oxidative stress, dependant on the prion status of cells. In contrast, localisation of three control mRNAs (CDC19, SUP45, DHH1) was unaffected by oxidative stress. RNQ1 and SUP35 foci were found to overlap with markers of P-Bodies and stress granules, with SUP35 having a particularly strong overlap with stress granules. Deletion of a stress granule component, Ubp3p, ameliorated the changes in localisation of the two mRNAs following oxidative stress. Taken together, these data suggest that the number of RNQ1 and SUP35 mRNA foci increases in response to oxidative stress due to increased localisation to stress granules. RNQ1, and to a lesser extent SUP35, mRNAs were additionally found to colocalise with aggregates of the Rnq1p protein. This suggests that Sup35p and Rnq1p nascent proteins may become exposed to [PIN+] immediately following translation and thus susceptible to templating to form their prion forms. The biological function of Rnq1p is unknown, so transcriptomics was used to investigate the possible functions of Rnq1p in both its soluble and prion forms. Widespread transcriptional changes were identified in strains with varying prion status indicating that [PSI+] and [PIN+] prions have large effects on gene expression. In contrast, loss of Rnq1p caused minimal transcriptional changes apart from to the expression of key chaperones including sequestrases (HSP42, BTN2) which may indicate a potential role for Rnq1p in protein homeostasis. Taken together, the results presented in this thesis point to new links between prion formation, protein localisation and oxidative stress induced mRNP granule formation.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Christopher Grant (Supervisor) & Mark Ashe (Supervisor)