DISSERTATION Titel der Dissertation Molecular Pathology of Mitochondrial tRNAIle Mutations Verfasserin Dipl.-Biol. Andrea Fettermann angestrebter akademischer Grad Doktorin der Naturwissenschaften (Dr. rer. nat.) Wien, 2012 Studienkennzahl lt. Studienblatt: A 091 441 Dissertationsgebiet lt. Studienblatt: Genetik - Mikrobiologie Betreuerin / Betreuer: Univ.-Prof. Dr. Andrea Barta Contents CONTENTS Contents………………………………………………………………………….………....... I Abbreviations……………………………………………………………...….…...……….... V Abstract……………………………………….………………………...…….….……......… 1 Zusammenfassung…………………………………………………...……….…..…..……. 3 1. Introduction .......................................................................................................... 5 1.1 Mitochondria ..................................................................................................... 5 1.1.1 Organization of Mitochondria ...................................................................... 5 1.1.2 Functions of Mitochondria .......................................................................... 6 1.1.3 The Respiratory Chain ................................................................................ 7 1.1.3.1 Complex I: NADH:Ubiquinone Oxidoreductase .................................... 8 1.1.3.2 Complex II: Succinate:Ubiquinone Oxidoreductase ............................. 8 1.1.3.3 Complex III: Ubiquinone-Cytochrome c Oxidoreductase ..................... 9 1.1.3.4 Complex IV: Cytochrome c Oxidase .................................................... 9 1.1.3.5 Complex V: ATP Synthase .................................................................10 1.1.4 Reactive Oxygen Species (ROS) ..............................................................10 1.1.5 The Mitochondrial Genome .......................................................................11 1.1.6 Features of Mitochondrial Genetics ...........................................................13 1.1.7 Transcription, Processing and Translation ................................................14 1.2 Mitochondrial tRNAs ........................................................................................17 1.2.1 tRNA Function ...........................................................................................17 1.2.2 tRNA Structure ..........................................................................................18 1.2.2.1 Primary Structure ................................................................................18 1.2.2.2 Secondary Structure ...........................................................................19 1.2.2.3 Tertiary Structure ................................................................................20 1.2.3 Biogenesis of tRNA ...................................................................................21 1.2.3.1 5’ Processing ......................................................................................21 1.2.3.2 3’ Processing ......................................................................................22 1.2.3.3 Posttranscriptional Modifications .........................................................23 1.2.3.4 CCA Adding ........................................................................................23 I Contents 1.2.4 tRNAIle .......................................................................................................24 1.3 Mitochondriopathies .........................................................................................24 1.3.1 Nuclear DNA Mutations .............................................................................25 1.3.2 Mitochondrial DNA Mutations ....................................................................26 1.3.2.1 Homoplasmic Mutations ......................................................................28 1.3.2.2 Heteroplasmic Mutations ....................................................................29 1.3.3 tRNA Mutations .........................................................................................30 1.3.4 tRNAIle Mutations .......................................................................................31 1.3.5 Model Systems ..........................................................................................34 1.3.5.1 The Cybrid Model ................................................................................35 2. Aim of the Project ...............................................................................................37 3. Case Reports .....................................................................................................38 3.1 m.4281A>G ......................................................................................................38 3.2 m.4284G>A ......................................................................................................39 3.3 m.4290T>C ......................................................................................................40 3.4 m.4296G>A ......................................................................................................40 3.5 m.4300A>G ......................................................................................................41 4. Results ...............................................................................................................43 4.1 Cellular and Biochemical Phenotype of 143B Cybrid Cells ..............................43 4.1.1 Cell Growth in Galactose Medium .............................................................44 4.1.2 Polarographic Analysis of Cell Respiration ................................................46 4.1.3 Spectrophotometric Analysis of Respiratory Chain Enzyme Activity .........49 4.1.4 mtDNA Copy Number ...............................................................................50 4.1.5 Sequencing of mtDNA ...............................................................................51 4.1.6 Structure of Mitochondria ..........................................................................53 4.2 Effects of the tRNAIle Mutations at the Molecular Level ....................................56 4.2.1 Steady-State Levels of tRNAIle ..................................................................56 4.2.2 Steady-State Levels of Precursor tRNAIle ..................................................57 4.2.3 Processing of the tRNAIle Precursor in vitro ...............................................60 4.2.4 tRNAIle Stability and Resynthesis ..............................................................61 4.2.5 tRNAIle Aminoacylation in Cybrid Cells ......................................................64 4.2.6 tRNAIle Aminoacylation “in Organello” .......................................................65 4.2.7 Mitochondrial Protein Synthesis ................................................................68 II Contents 4.2.8 Steady-State Levels of Mitochondrial Proteins ..........................................68 4.2.9 Secondary Structure Prediction of tRNAIle .................................................71 5. Discussion ..........................................................................................................75 5.1 Aim of the Study ...............................................................................................75 5.2 The Cybrid Model .............................................................................................75 5.3 Characterization of the Molecular and Cellular Phenotypes of Individual Mutations ...............................................................................................................77 5.3.1 m.4281A>G ...............................................................................................77 5.3.2 m.4284G>A ...............................................................................................82 5.3.3 m.4290T>C ................................................................................................85 5.3.4 m.4296G>A ...............................................................................................90 5.3.5 m.4300A>G ...............................................................................................94 5.3.6 m.4263A>G ...............................................................................................97 5.4 Molecular Pathogenesis in Comparison ...........................................................97 6. Materials & Methods .........................................................................................105 6.1 Cell Culture ....................................................................................................105 6.1.1 Passaging, Freezing and Unfreezing of Cells ..........................................106 6.1.2 Cell Counting ...........................................................................................106 6.1.3 Growth Assays ........................................................................................106 6.2 Miscellaneous Cell Treatment ........................................................................107 6.2.1 Mitotracker Staining .................................................................................107 6.2.2 Electron Microscopy ................................................................................107 6.3 Biochemistry: Respiratory Chain Activity ........................................................107 6.3.1 Polarography: Cell Respiration ................................................................107 6.3.2 Spectrophotometry: Respiratory Chain Complex Activities .....................108