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Ribosome Assembly, Proteolysis and Pathogenesis in Human Mitochondria

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Ribosome Assembly, Proteolysis and Pathogenesis in Human Mitochondria Ribosome Assembly, Proteolysis and Pathogenesis in Human Mitochondria Christie L Waddington Wellcome Trust Centre for Mitochondrial Research Institute for Neuroscience Newcastle University Thesis submitted to Newcastle University in candidature for the degree of Doctor of Philosophy September 2017 2 ABSTRACT The human mitochondrial genome is approximately 16.6kbp and encodes 22 tRNAs, 2 rRNAs and 13 polypeptides. These polypeptides are synthesised by the mitoribosome, composed of a large (39S) and small (28S) subunit. Although recently published cryo-EM structures of the human, porcine and yeast mitoribosomes have identified a number of significant differences including new components with uncharacterised functions. One such component was mS38 (AURKAIP1) as a part of the mt-SSU of the mitoribosome. This was consistent with previous work in my host laboratory using a siRNA screen of candidate mitochondrial proteins, which identified AURKAIP1 as having a role in mitochondrial gene expression. I continued this work and confirmed levels of AURKAIP1 are tightly controlled within the cell. Overexpression of both AURKAIP1 and AURKAIP1-FLAG caused disruption to the mitoribosome and mitochondrial translation, and depletion of AURKAIP1 impaired cell growth and resulted in cell death. Although the exact function of AURKAIP1 is still elusive, AURKAIP1 may have a role as an RNA chaperone. When overexpressed, AURKAIP1 was cleaved by the mitochondrial matrix protease LONP1, from a 25kDa to a 15kDa species by two cleavage events, although this could not be determined in the endogenous species. This work led to the characterisation of LONP1 variants that were believed to be pathogenic. Whole exome sequencing identified two novel mutations (p.Tyr565His; p.Glu733Lys) in LONP1 in a paediatric patient with a severe mitochondrial disease. By cloning and sequencing LONP1 from patient samples, I showed compound heterozygosity in the patient for 2 independent mutations, confirming the disorder to be autosomal recessive. Skeletal muscle showed severe defects in OXPHOS complexes I, III and IV, reduced mtDNA copy number and a decrease in the steady state levels of TFAM. Steady state levels of LONP1 remained unaffected. The p.Tyr565His mutation is localised to a highly conserved aromatic- hydrophobic (Ar-φ) motif present in hexameric ATP-dependent proteases and in vitro assays indicated a retained ability to hydrolyse ATP but a loss of proteolytic activity. This suggests that the mutation is likely to prevent the substrate from reaching the proteolytic site. i ii ACKNOWLEDGEMENTS First and foremost, I would like to give my uttermost thanks to my supervisors Prof. Rob Taylor, Prof. Zofia Chrzanowska-Lightowlers and Prof. Robert Lightowlers for the giving me the opportunity to pursue this PhD project. Their continued support and guidance has allowed me to develop both personally and professionally and for that I will always be grateful. Secondly, I would also like to thank Professor Maria Falkenberg and her colleagues at Gothenburg University for welcoming me into their lab and sharing their expertise. I am eternally grateful to Dr Jeremy Brown for being a great mentor over the last 7 years, having played such a key part in my scientific and personal development. I would like to thank everyone in the MRG and NHS Highly Specialised Mitochondrial Diagnostic Service who have given me technical and emotional support throughout my PhD. My project has been a collaborative effort and I have been very fortunate to work with a group of people who have been willing to give up their time and expertise when I have needed. I am truly going to miss working in such a great environment. Thanks also go to lab members past and present: Kyle, Monika, Ewen, Nicole, Rawaa, Marysia, Francesco, Yasmin and particularly to my office girls Fei and Agata, whose friendship and advice over the last 4 years have been indispensable. I would like to thank Saltwell Harriers, especially the lads, who have welcomed me into the club and have provided great respite over the course of my studies. I look forward to running with you for many years to come as Lady W. I would like to thank my family and friends who have supported me over the years, particularly to my father Ian Waddington who sadly passed away in December 2016. He allowed me to pursue my love of sport, my love of learning and was always there to offer advice. And finally, I would like to thank Dr Robert Panting, who supported me throughout this journey. iii AUTHOR’S DECLARATION This thesis is submitted for the degree of Doctor of Philosophy at Newcastle University. The research was conducted at the Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, under the supervision of Professor Robert N. Lightowlers, Professor Zofia M.A. Chrzanowska-Lightowlers and Professor Robert W. Taylor. All research is my own unless stated otherwise. I certify that none of the material offered in this thesis has been previously submitted by me for a degree of any other qualification at this, or any other, university. iv TABLE OF CONTENTS Abstract i Acknowledgements iii Author’s Declaration iv List of Figures x List of Tables xiii Abbreviations and Symbols xiv CHAPTER 1: INTRODUCTION 1 1.1 – Mitochondria: An Overview 1 1.1.1 Evolutionary Origin of Mitochondria 1 1.1.2 Structure of Mitochondria 2 1.1.3 Mitochondrial Dynamics 5 1.1.4 The OXPHOS System 6 1.1.5 Additional Functions of Mitochondria 8 1.2 – Mitochondrial DNA 10 1.2.1 Replication 11 1.2.2 Transcription 12 1.2.3 Post-transcriptional Modifications 12 1.3 – Mitochondrial Protein Synthesis 13 1.3.1 The Mitoribosome 14 1.3.2 Mitoribosome Assembly 15 1.3.3 Initiation 17 1.3.4 Elongation 18 1.3.5 Termination 19 1.3.6 Ribosome Recycling 20 1.4 – Nuclear-Encoded Mitochondrial Proteins 20 1.4.1 Protein Import 20 1.4.2 Identification of Nuclear-Encoded Mitochondrial Proteins 22 1.4.3 AURKAIP1: mS38 22 1.5 – Mitochondrial Proteases 25 1.5.1 LONP1 28 1.6 – Mitochondrial Diseases 29 1.7 – Aims of the Project 32 CHAPTER 2: MATERIALS AND METHODS 33 2.1 – Cell Culture 33 2.1.1 Mammalian Cell Lines and Tissues 33 v 2.1.2 Cell Culture Maintenance 34 2.1.3 Cell Counting 34 2.1.4 Cell Line Storage and Thawing 34 2.1.5 Mycoplasma Testing 35 2.1.6 Transfection with siRNA 35 2.1.7 Stable Transfection 36 2.1.8 Expression of Recombinant Protein in Mammalian Cell Lines 36 2.2 – Bacterial Manipulation 36 2.2.1 Bacterial Culture and Storage 36 2.2.2 Transformation 37 2.2.3 Expression of Recombinant Protein in Bacterial Cell Lines 37 2.2.4 Bacterial Cell Lysis 37 2.2.5 Cracking Gel Bacterial Lysis 38 2.3 – DNA Manipulation 38 2.3.1 Isolation of Plasmid DNA from Bacterial Cultures 38 2.3.2 Restriction Digest 38 2.3.3 Dephosphorylation of Vectors 38 2.3.4 Ligation 39 2.3.5 Phenol/Chloroform Extraction 39 2.3.6 Ethanol Precipitation 39 2.3.7 Measuring Nucleic Acid Concentration 39 2.3.8 DNA Extraction from Whole Cells 40 2.3.9 Agarose Gel Electrophoresis 40 2.3.10 Polymerase Chain Reaction (PCR) 40 2.3.11 Real-Time PCR (qPCR) 41 2.3.12 Sanger Sequencing 41 2.3.13 Whole Exome Sequencing, Analysis and Interpretation 41 2.4 – RNA Manipulation 42 2.4.1 RNA Extraction from Whole Cells 42 2.4.2 Reverse Transcription 42 2.4.3 Ammonium Acetate Precipitation 42 2.4.4 Denaturing Agarose Gel Electrophoresis 43 2.4.5 High Resolution Northern Blot 43 2.5 – Protein Manipulation 45 2.5.1 Cell Lysis 45 2.5.2 Isolation of Mitochondria from Mammalian Cell Cultures 45 2.5.3 Mitochondrial Shaving 46 2.5.4 Total Protein Extraction from Muscle Tissue 46 2.5.5 SDS-PAGE 46 2.5.6 Coomassie Brilliant Blue Staining 47 2.5.7 Silver Staining 47 2.5.8 Western Blot and Immunodetection 47 2.5.9 Bradford Assay 48 2.5.10 Isokinetic Sucrose Gradient Separation 48 2.5.11 Trichloroacetic Acid (TCA) Precipitation of Proteins 49 vi 2.5.12 Immunoprecriptation (IP) 49 2.5.13 Protein Purification using Nickel Resin 50 2.5.14 Protein Purification using Heparin Sepharose 51 2.5.15 Protein Purification using Q Sepharose 52 2.5.16 Gel Filtration analysis 52 2.5.17 Microscale Thermophoresis (MST) 53 2.5.18 Thermofluor Stability Assay 53 2.6 – In Vitro Protein Import Assay 54 2.6.1. In Vitro Transcription 54 2.6.2. In Vitro Translation and Analysis 54 2.6.3. Isolation of Rat Liver Mitochondria 55 2.6.4. In Vitro Protein Import 55 2.7 - In Vitro Protein Activity Assays 56 2.7.1. In Vitro ATPase assay 56 2.7.2. In Vitro TFAM Degradation assay 57 CHAPTER 3: LEVELS OF AURKAIP1 ARE TIGHTLY CONTROLLED 59 3.1 – Introduction 59 3.2 – Results 61 3.2.1 Expression of AURKAIP1 and AURKAIP1-FLAG in Flp-In™ TREx™ cells 61 3.2.2 Effects of AURKAIP and AURKAIP1-FLAG overexpression 61 3.2.3 Effects of depletion in mammalian cell lines 64 3.3 – Discussion 72 CHAPTER 4: IS AURKAIP1 A COMPONENT OF THE MATURE HUMAN MITORIBOSOME? 75 4.1 – Introduction 75 4.2 – Results 77 4.2.1 Overexpressed AURKAIP1 does not associate with the mitoribosome 77 4.2.2 AURKAIP1-FLAG associates with mitoribosomal RNA species 82 4.2.3 Mycoplasma stress increases levels of a mitochondrial protein 85 4.3 – Discussion 95 CHAPTER 5: DETERMINING THE TRUE SIZE OF AURKAIP1 101 5.1 – Introduction 101 5.2 – Results 104 5.2.1 Determining the cleavage of AURKAIP1 in vitro 104 5.2.2 Optimising in vitro translation 106 5.2.3 Depletion of matrix proteases 111 5.2.4 Novel AURKAIP1 species do not associate with the mitoribosome 114 5.3 – Discussion 116 vii CHAPTER 6: INVESTIGATING A PATIENT WITH MUTATIONS IN LONP1 121 6.1 – Introduction 121 6.2 – Results 123 6.2.1 Identifying a patient with LONP1 mutations
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