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Role of Mrna Surveillance Pathways During Oxidative Stress in Saccharomyces Cerevisiae a Thesis Submitted to the University of M

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Role of Mrna Surveillance Pathways During Oxidative Stress in Saccharomyces Cerevisiae a Thesis Submitted to the University of M Role of mRNA surveillance pathways during oxidative stress in Saccharomyces Cerevisiae A thesis submitted to The University of Manchester for the degree of DOCTOR OF PHILOSOPHY in the Faculty of Biology, Medicine and Health 2017 NUR HIDAYAH JAMAR SCHOOL OF BIOLOGICAL SCIENCES 1 Table of Content Table of Content 2 List of Figures 9 List of Tables 12 Declaration 13 Copyright statement 13 Communications 14 Publication 14 Contributor’s acknowledgment 15 Acknowledgments 16 List of abbreviations 17 Abstract 21 1.0 introduction 23 1.1 Generation of reactive oxygen species (ROS) 23 1.2 Sources of ROS and commonly used ROS compounds 25 1.3 What happens when cells cannot handle oxidative stress? 26 1.3.1 Lipid peroxidation 27 1.3.2 Protein oxidation 29 1.3.3 Oxidatively damaged nucleic acids (DNA and RNA) 30 1.4.Transcriptional responses of S. cerevisiae during oxidative stress 31 conditions 1.4.1 Regulation of gene expression by Yap1 32 2 1.4.2 Modulation of the general stress response by MSN2/MSN4 33 1.5 Translational responses of S. cerevisiae to oxidative stress conditions 34 1.5.1 Overview of protein synthesis 34 1.5.1.1 Translation initiation 34 1.5.1.2 Translation elongation 37 1.5.1.3 Translation termination 39 1.5.2 Regulation of translation initiation during oxidative stress in S. 39 cerevisiae 1.5.2.1 Regulation of TC by eIF2α 41 1.5.2.2 Regulation of mRNA-specific translational control via Gcn4 43 1.6 Cytoplasmic mRNA degradation in S. cerevisiae 43 1.6.1 Normal mRNA degradation 44 1.6.2 Specialized mRNA quality control mechanisms 47 1.6.2.1 Nonsense-mediated decay (NMD) 48 1.6.2.2 Nonstop mRNA decay (NSD) 51 1.6.3.3 No-go mRNA decay (NGD) 53 1.7 Protein folding, misfolding, and aggregation 55 1.7.1 Amyloid aggregates 57 1.7.2 Amorphous aggregates 58 1.7.3 Protein aggregation related diseases 59 1.8 Prions 60 1.8.1 Mammalian prions 61 1.8.2 Yeast prions 64 1.8.2.1 [PSI+] 65 1.8.2.2 [PIN+] 67 1.9 Thesis objectives 68 3 2.0 Materials and Methods 69 2.1 Yeast strains and plasmids 69 2.1.1 Yeast strains 69 2.1.2 Plasmids 70 2.2 Strain construction and verification 71 2.2.1 Oligonucleotides 71 2.2.2 DNA amplification by polymerase chain reaction (PCR) 73 2.2.3 Yeast genomic DNA extraction 73 2.3 DNA/RNA manipulation and analysis 74 2.3.1 Plasmid extraction 74 2.3.2 Yeast transformation 75 2.3.3 Agarose gel electrophoresis 76 2.3.4 Quantitative Reverse Transcriptase PCR (qRT-PCR) 76 2.3.4.1 RNA extraction 77 2.3.4.2 RNA washes 77 2.3.4.3 q-RT PCR analysis 77 2.3.5 Quantitation of DNA/ RNA concentrations using a spectrophotometer 78 2.4 Media and growth conditions 78 2.4.1 S. cerevisiae 78 2.4.2 E. coli 79 2.5 Yeast growth and stress analysis 79 2.5.1 Spot tests for oxidant-stress sensitivity 79 2.5.2 Oxidant-growth sensitivity 79 2.5.3 Cell viability assay 80 2.6 Protein Analysis 80 2.6.1 Polysome analysis 80 4 2.6.1.1 Preparation of yeast cell extracts for polysome analysis 80 2.6.1.2 Preparation of sucrose gradients 81 2.6.1.3 Sedimentation of polyribosomes 82 2.6.2 35S cysteine/methionine radiolabelling 83 2.6.3 β-galactosidase reporter assays to measure stop codon readthrough 84 2.6.3.1 Preparation of whole cell extracts 84 2.6.3.2 Assay 84 2.6.4 Dual-luciferase reporter assays to measure stop codon readthrough 85 2.6.4.1 Preparation of whole cell extracts 85 2.6.4.2 Assay 86 2.6.5 Western blot analysis 86 2.6.5.1 Preparation of yeast whole cell extracts 86 2.6.5.2 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis 87 (SDS-PAGE) and western blotting 2.7 Analysis of protein aggregation 89 2.7.1 Preparation of yeast cell cultures 89 2.7.2 Silver-staining of SDS-PAGE gels 90 2.7.3 Mass spectrometry 90 2.7.4 Bioinformatic analysis of aggregated proteins 90 2.8 Live-cell fluorescence microscopy 91 2.8.1 Preparation of yeast samples 91 2.8.2 DeltaVision fluorescence microscopy 91 2.9 Analysis of prion formation 91 2.9.1 Determination of de novo [PSI+] prion formation 92 2.9.2 Determination of de novo [PIN+] Prion Formation 93 5 3.0 Importance of mRNA surveillance pathways during 94 oxidative stress conditions 3.1 Introduction 94 3.2 Results 96 3.2.1 Construction of mRNA surveillance mutants 96 3.2.2 Phenotypic assays of [psi-] and [PSI+] strains 99 3.2.3 Requirement for mRNA surveillance pathways during oxidative stress 99 3.2.4 Growth and viability analysis of mRNA surveillance mutants during 100 oxidative stress conditions 3.2.4.1 The oxidant sensitivity of ski8 mutant depends on its [PSI+] 102 status 3.2.4.2 Loss of NMD may act to improve oxidant tolerance and this 104 phenotype is further observed in [PSI+] background 3.2.4.3 Mutants in the NGD pathway are modestly sensitive to 104 oxidative stress 3.2.5 Analysis of translational activity in mRNA surveillance mutants 106 during oxidative stress conditions 3.2.6 Analysis of protein synthesis in mRNA surveillance mutants during 108 oxidative stress conditions 3.3 Discussion 114 4.0 Requirement for nonstop decay during oxidative stress 116 conditions 4.1 Introduction 116 4.2 Results 118 4.2.1 Phenotypic assays of ski mutants 118 4.2.2 The SKI complex is required for oxidant tolerance 118 6 4.2.3 Analysis of NSD in ski complex and ski7 mutants 122 4.2.4 Overlapping requirements for Ski7 and Dom34/Hbs1 during oxidative 124 stress conditions 4.2.5 Oxidative stress Sup35 aggregation in dose-dependent manner and 128 potentially generates NSD substrate 4.2.6 Overexpression of Sup35 rescues oxidant sensitivity in WT and SKI 131 complex mutant strains 4.2.7 Overexpression of Sup35 decreases stop codon readthrough during 133 oxidative stress conditions in WT and ski2 mutant strains 4.3 Discussion 137 5.0 Importance of mRNA quality control for proteostasis 143 5.1 Introduction 143 5.2 Results 145 5.2.1 Loss of mRNA surveillance mutants causes widespread protein 145 aggregation 5.2.2 More proteins are susceptible to aggregation following loss of mRNA 147 surveillance mutant strains 5.2.3 Differences in subcellular localization of proteins isolated from 148 aggregates in mRNA surveillance mutants 5.2.4 Enrichment of functional categories within protein aggregates isolated 150 from mRNA surveillance mutants 5.2.5 Analysis of the physicochemical properties of aggregated proteins 152 identified in mRNA surveillance mutants 5.2.5.1 Aggregated proteins are enriched for abundant and highly 153 expressed proteins 5.2.5.2 Aggregated proteins are more hydrophobic than the 155 unaggregated ones 5.2.6 Analysis of selected amino acid composition in mRNA surveillance 157 mutant aggregates 7 5.2.7 Molecular chaperones are present within protein aggregates 159 5.2.8 An increased frequency of de novo prion formation occurs in NMD 161 mutants 5.2.9 The frequency of induced [PSI+] formation is increased in NMD 164 mutants 5.2.10 The [PSI+] status of strains improves cell viability upon exposure to 166 various stress conditions 5.3 Discussion 170 6.0 General discussion 177 6.1 Possible interaction between NGD complex and Gcn2 kinase 181 6.2 Aggregation of Sup35 upon oxidative stress: where does it take us? 182 6.3 Amorphous aggregates may potentially be more toxic than amyloid 183 aggregates 6.4 The presence of 22 PTCs in strain 74D-694 may actually contribute to 184 increased tolerance to various stresses particularly in NMD mutants 7.0 Bibliography 188 8.0 Appendix 201 Appendix 1 List of protein aggregates present within WT strain 201 Appendix 2 List of protein aggregates present within ski7 mutant 202 Appendix 3 List of protein aggregates present within ski8 mutant 204 Appendix 4 List of protein aggregates present within upf2 mutant 206 Appendix 5 List of protein aggregates present within hbs1 mutant 208 8 List of Figures Figure 1.1 Mechanism of ROS production and conversion 24 Figure 1.2 Mechanism of lipid peroxidation 28 Figure 1.3 Simplified overview of the translation initiation process in 35 eukaryotes Figure 1.4 Mechanism of translation elongation 38 Figure 1.5 Mechanism of translation termination in yeasts 40 Figure 1.6 Regulation of translation initiation by inhibiting TC formation 4 2 Figure 1.7 Normal mRNA decay mechanisms in eukaryotes 45 Figure 1.8 Recognition of NMD substrates 49 Figure 1.9 Recognition of NSD substrates 53 Figure 1.10 Recognition of NGD substrates 55 Figure 1.11 Comparison of the mechanisms between mammalian and yeast 62 [PSI+] prion formations Figure 2.1 An example of a normal, untreated polysome trace from actively 83 translating yeast cells Figure 3.1 Construction of mRNA surveillance mutants in strain 74D-694 97 using HIS3 deletion cassette Figure 3.2 Construction of mRNA surveillance mutants in strain 74D-694 98 using HIS3 deletion cassette Figure 3.3 Oxidative stress-sensitivity of mRNA surveillance mutants 101 Figure 3.4 ski8 mutant is hypersensitive to oxidative stress conditions but 103 this sensitivity is lost in the [PSI+] version Figure 3.5 The oxidant tolerance of NMD mutants depends on their [PSI+] 105 status Figure 3.6 Mutants in NGD pathway are modestly sensitive to oxidative 107 9 stress Figure 3.7 Differences in oxidant sensitivity do not arise due to translational 109 activity in NSD mutants Figure 3.8 NMD mutants exhibit moderate effect of inhibition of translation 110 initiation Figure 3.9 Inhibition of translation initiation was observed in NGD mutants 111 even in unstressed condition Figure 3.10 Hydrogen peroxide causes inhibition of protein synthesis all 113 strains following 1 hour treatment Figure 4.1 Mutants in the Ski complex are sensitive to oxidative stress 119 Figure 4.2 Mutants in the Ski complex are sensitive to oxidative stress 121
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