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Université de Strasbourg 2013 Ecole doctorale des Sciences de la Vie et de la Santé THÈSE présentée pour l’obtention du grade de DOCTEUR DE L’UNIVERSITE DE STRASBOURG Discipline : Sciences du Vivant Domaine : Aspects moléculaires et cellulaires de la biologie par GOWHER Ali Characterization of protein factors targeting RNA into human mitochondria Soutenue le 17 September 2013 devant le jury de thèse composé comme suit: Professor Andre Schneider Rapporteur externe Dr Anne Lombès Rapporteur externe Dr Phillipe Giege Examinateur interne Dr Ivan Tarassov Directeur de thèse UMR N°7156 UdS-CNRS «Génétique Moléculaire, Génomique et Microbiologie» TABLE OF CONTENTS TABLE OF CONTENTS TABLE OF CONTENTS LIST OF FIGURES ACKNOWLEDGEMENTS ABBREVIATIONS 1. Introduction...................................................................................................................1! 1.1. Origin and structure of mitochondria.......................................................................1! 1.2. Function of mitochondria.........................................................................................3! 1.3. Human mitochondrial genome.................................................................................5! 1.3.1. Structure and composition................................................................................5! 1.3.2. Nucleoid organization.......................................................................................7! 1.4. Human mtDNA replication......................................................................................8! 1.4.1. Proteins participating in mtDNA replication....................................................8! 1.4.2. Mechanism of mtDNA replication..................................................................10! 1.4.3. Initiation of Heavy strand replication.............................................................12! 1.4.4. Initiation of light strand replication................................................................13! 1.5. Human mtDNA transcription.................................................................................14! 1.5.1. Transcription machinery.................................................................................14! 1.5.2. Transcription of mitochondrial DNA..............................................................15! 1.5.3. Termination of transcription...........................................................................16! 1.5.4. Processing of RNA transcript.........................................................................17! 1.6. Translation of mitochondrial DNA........................................................................17! 1.6.1. Translation machinery....................................................................................18! 1.6.2. Mitochondrial translation process...................................................................19! 1.7. Macromolecular import into mitochondria............................................................20! 1.7.1. Mitochondrial protein import..........................................................................20! 1.7.2. Mitochondrial targeting and sorting signals...................................................20! 1.7.3. Mitochondrial protein import machinery........................................................22! 1.7.3.1. The TOM complex...................................................................................22! TABLE OF CONTENTS 1.7.3.2. The TIM23 complex................................................................................23! 1.7.3.3. SAM complex..........................................................................................25! 1.7.3.4. The MIA pathway....................................................................................26! 1.7.3.5. TIM22 complex.......................................................................................27! 1.8. Nucleic acid import................................................................................................29! 1.8.1. DNA import into mitochondria.......................................................................29! 1.8.2. RNA import into mitochondria.......................................................................29! 1.8.2.1. RNA import into protozoans mitochondria.............................................30! 1.8.2.2. RNA import into Plant mitochondria.......................................................33! 1.8.2.3. RNA import in Yeast mitochondria.........................................................35! 1.8.2.4. RNA import in mammals.........................................................................39! 2. Results..........................................................................................................................49! 2.1. Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase.................................................................................49! 2.1.1. Summary.........................................................................................................49! 2.1.2. Publication 1..................................................................................................52! 2.2. Human enolases facilitate tRK1-preKARS2 complex formation..........................53! 2.3. Mutation in PNPT1, which Encodes a Polyribonucleotide Nucleotidyltransferase, impairs RNA import into mitochondria and causes respiratory chain deficiency..56! 2.3.1. Summary.........................................................................................................56! 2.3.2. Publication 2..................................................................................................58! 2.4. PNPase overexpression increased RNA import into mitochondria.......................59! 2.5. PNPase down-regulation decreased RNA import into mitochondria....................61! 2.6. Identification of proteins interacting with PNPase................................................62! 3. Discussion....................................................................................................................59! 3.1. preKARS2 interacts with yeast tRK1 and its derivatives......................................59! 3.2. Human enolases facilitate tRK1-preKARS2 complex formation..........................60! 3.3. preKARS2 as a tRK1 carrier into human mitochondria........................................61! 3.4. Mitochondrial import of small truncated RNAs....................................................62! 3.5. PNPase translocate RNA into the mitochondrial matrix.......................................63! 3.6. PNPase mutation affect mitochondrial translation................................................64! TABLE OF CONTENTS 3.7. Divergent mechanisms for convergent goal..........................................................65! 4. Conclusions and perspectives....................................................................................73! 5. Materials and Methods...............................................................................................76! 5.1. Materials................................................................................................................76! 5.1.1. Bacterial strains...............................................................................................76! 5.1.2. Human cell lines.............................................................................................76! 5.1.3. Plasmids and oligonucleotides........................................................................77! 5.2. Methods.................................................................................................................78! 5.2.1. Electroporation of E. coli................................................................................78! 5.2.2. Plasmid DNA preparation...............................................................................78! 5.2.3. Recombinant protein purification...................................................................78! 5.2.4. Polymerase chain reaction (PCR)...................................................................79! 5.2.5. RNA synthesis................................................................................................80! 5.2.6. Radioactive labeling and aminoacylation.......................................................80! 5.2.7. Radioactive labeling of oligonucleotides probes............................................81! 5.2.8. Electrophoretic mobility shift assay (EMSA).................................................81! 5.2.9. Cell culture......................................................................................................82! 5.2.10. Transfection of human cell lines...................................................................83! 5.2.10.1. Transfection with T7 transcript..............................................................83! 5.2.10.2. Transient transfection with plasmid.......................................................84! 5.2.10.3. Transient protein downregulation..........................................................84! 5.2.11. Purification of mitochondria.........................................................................85! 5.2.12. In vitro import assay.....................................................................................86! 5.2.13. Northern hybridization..................................................................................86! 5.2.14. Immunoblotting.............................................................................................87! 5.2.15. Mitochondrial translation analysis in vivo....................................................88! 5.2.16. North-western blot........................................................................................88! 5.2.17. Cross linking immuno precipitation (CLIP).................................................89! 5.2.18. Mass Spectromery Analysis..........................................................................89 BIBLIOGRAPHY........................................................................................................................91 RÉSUMÉ DU THÈSE...............................................................................................................118 LIST OF FIGURES Figure 1: Mitochondrion.................................................................................................................2! Figure 2: Schematic of the mitochondrial electron transport chain................................................4! Figure 3: The human mitochondrial genome..................................................................................6! Figure 4: Mitochondrial DNA replication machinery ...................................................................8! Figure 5: Models of mitochondrial DNA replication...................................................................11! Figure 6: Schematic representation of the D-loop regulatory region...........................................13! Figure 7: Targeting and Sorting Signals of mitochondrial precursor proteins.............................21! Figure 8: The presequence pathway of protein import into mitochondria....................................23! Figure 9: Sorting and assembly machinery (SAM) of outer mitochondrial membrane...............26! Figure 10: The machinery for import and assembly (MIA) of preprotein into mitochondrial intermembrane space....................................................................................................................27! Figure 11: The carrier pathway of preprotein import into mitochondria......................................28! Figure 12: Overview of mitochondrial tRNA-import channels....................................................34! Figure 13: Lysine tRNAs in S. cereviseae....................................................................................36! Figure 14: Mitochondrial tRNA import in S. cerevisiae...............................................................37! Figure 15: Structure of Human 5S rRNA.....................................................................................42! Figure 16: Hypothetical mechanism of 5S rRNA intracellular traffic..........................................44! Figure 17: Predicted structures of the yeast tRNALys(CUU) and small synthetic RNAs...............46! Figure 18: Protein purification and electrophoretic mobility shift assay......................................53! Figure 19: Interaction between labeled tRK1 and preKARS2 in the absence or presence of human enolases.............................................................................................................................55! Figure 20: Implication of PNPase in mitochondrial RNA import in vivo....................................60! Figure 21: Implication of PNPase in mitochondrial RNA import in vivo....................................62! ACKNOWLEDGEMENTS First of all, with utmost pleasure, I express my heartfelt and profound gratitude to Dr. Ivan Tarassov, my PhD supervisor, whose guidance, unflinching support and valuable feedback made this work possible. Thank you Ivan, for giving me the opportunity to join your lab, for introducing me to the field of mitochondrial research, for administrative and financial support during my 4th year of PhD, for your kind scientific assistance and for the great moments I passed in your garden parties. I am highly indebted to Dr. Nina Entelis, my additional supervisor, for her excellent continuous support and guidance throughout my PhD. I am thankful to you Nina, for your kind advices and assistance, for your personal scientific interests in my work, and for your help in interpreting my experimental results on daily basis. I am extremely grateful to Prof. André Schneider, Dr. Anne Lombès, and Dr. Phillipe Giegé, for accepting to be the members of my thesis jury, for their precious time and efforts to evaluate my thesis. I am also thankful to Dr. Mikhail Vysokikh, a former postdoctoral fellow, who was my scientific guru during the last three years of my PhD. Mike you really were an invaluable help, and I want to thank you for all the scientific knowledge you taught me, for your brilliant assistance in every situation and for our close and fruitful cooperation. I wish you all the best for your bright future in Moscow. I would like to extend my gratitude to all other members of our “Mito” group, past and present, especially Dr Alexandre Smirnov “Sacha”, Anne-Marie Heckel, Yann Tonin, Dr Tom Schirtz, Dr Caroline Comte, and Ilya for creating a scientifically motivating environment, for all their help, support, suggestions and friendship. I also want to thank all our members of the UMR7156 for the great and very stimulating working atmosphere and for their kind help and discussions. I felt very happy among you during the past four years and I really enjoyed all our nice parties. I am grateful to Higher Education Commission (HEC) of Pakistan, SFERE (Société française d’exportation des resources éducatives), and Ivan Tarassov lab, for financing my study in France. Finally, I am thankful to all my family members who have stood alongside me all these years. Words are meaningless to what they have given me. It is, I believe their prayers and spiritual support, which were a constant source of strength and inspiration to me that despite, many constraints, I was able to complete my work. Gowher Ali ABBREVIATIONS ABBREVIATIONS ADP adenosine diphosphate ANT adenine nucleotide transporter ATAD3 ATPase family AAA domain-containing protein 3 ATP adenosine triphosphate BB breakage buffer BSA bovine serum albumin CLIP crosslinking combined to immunoprecipitation CSB conserved sequence block DEPC diethylpyrocarbonate DMEM dulbecco modified Eagle's medium DNA deoxyribonucleic acid Drp1 dynamin related protein 1 DTT dithiothreitol E.coli Escherichia coli EDTA ethylenediaminetetraacetic acid EMSA electrophoretic mobility shift assay Eno-2 enolase-2 FADH2 flavin adenine dinucleotide hydroquinone form FRET förster resonance energy transfer HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMG high mobility group HSP heavy strand promoter Hsp heat-shock protein ABBREVIATIONS IFs initiation factors IMS intermembrane space IPTG isopropyl ß-D-1-thiogalactopyranoside KDa kilo Dalton LB lysogeny broth LSP light strand promoter LSU large subunit Mfn mitofusin mtDNA mitochondrial DNA mtEFTu mitochondrial elongation factor Tu mtEFTs mitochondrial elongation factor Ts mtEFG mitochondrial elongation factor G mtRF mitochondrial release factor mtRPOL mitochondrial RNA polymerase mtSSB mitochondrial single stranded DNA binding protein MIA mitochondrial intermembrane space import and assembly MISS mitochondrial intermembrane space signal MMP mitochondrial processing peptidase MRPs mitochondrial ribosomal proteins MTS mitochondrial targeting sequence mRNA messanger RNA NADH nicotinamide adenine dinucleotide reduced OD optical density ABBREVIATIONS OPA1 optic atrophy 1 PAM presequence translocase associated motor PBS phosphate buffered saline PNPase polynucleotide phosphorylase POLG pitochondrial DNA polymerase ! PPR pentatricopeptide repeat RIC RNA import complex RITOLS RNA incorporation throughout lagging strand ROS reactive oxygen species rRNA ribosomal RNA PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction preMSK precursor of Saccharomyces cerevisiae mitochondrial lysyltRNA- synthetase preKARS2 precursor form of human mitochondrial lysyltRNA-synthetase SAM sorting and assembly machinery S.cerevisiae Saccharomyces cerevsisae SDS sodium dodecylsulfate SELEX systematic Evolution of Ligands by Exponential Enrichment siRNA small interferring RNA SSC saline sodium citrate buffer ssDNA single stranded DNA TAB tubulin antisense binding protein TAS termination associated sequence

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Université de Strasbourg. 2013. Ecole doctorale des Soutenue le 17 September 2013 devant le jury de thèse composé comme suit: Professor Andre
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Université de Strasbourg 2013 Ecole doctorale des Sciences de la Vie et de la Santé THÈSE présentée pour l’obtention du grade de DOCTEUR DE L’UNIVERSITE DE STRASBOURG Discipline : Sciences du Vivant Domaine : Aspects moléculaires et cellulaires de la biologie par GOWHER Ali Characterization of protein factors targeting RNA into human mitochondria Soutenue le 17 September 2013 devant le jury de thèse composé comme suit: Professor Andre Schneider Rapporteur externe Dr Anne Lombès Rapporteur externe Dr Phillipe Giege Examinateur interne Dr Ivan Tarassov Directeur de thèse UMR N°7156 UdS-CNRS «Génétique Moléculaire, Génomique et Microbiologie» TABLE OF CONTENTS TABLE OF CONTENTS TABLE OF CONTENTS LIST OF FIGURES ACKNOWLEDGEMENTS ABBREVIATIONS 1. Introduction...................................................................................................................1! 1.1. Origin and structure of mitochondria.......................................................................1! 1.2. Function of mitochondria.........................................................................................3! 1.3. Human mitochondrial genome.................................................................................5! 1.3.1. Structure and composition................................................................................5! 1.3.2. Nucleoid organization.......................................................................................7! 1.4. Human mtDNA replication......................................................................................8! 1.4.1. Proteins participating in mtDNA replication....................................................8! 1.4.2. Mechanism of mtDNA replication..................................................................10! 1.4.3. Initiation of Heavy strand replication.............................................................12! 1.4.4. Initiation of light strand replication................................................................13! 1.5. Human mtDNA transcription.................................................................................14! 1.5.1. Transcription machinery.................................................................................14! 1.5.2. Transcription of mitochondrial DNA..............................................................15! 1.5.3. Termination of transcription...........................................................................16! 1.5.4. Processing of RNA transcript.........................................................................17! 1.6. Translation of mitochondrial DNA........................................................................17! 1.6.1. Translation machinery....................................................................................18! 1.6.2. Mitochondrial translation process...................................................................19! 1.7. Macromolecular import into mitochondria............................................................20! 1.7.1. Mitochondrial protein import..........................................................................20! 1.7.2. Mitochondrial targeting and sorting signals...................................................20! 1.7.3. Mitochondrial protein import machinery........................................................22! 1.7.3.1. The TOM complex...................................................................................22! TABLE OF CONTENTS 1.7.3.2. The TIM23 complex................................................................................23! 1.7.3.3. SAM complex..........................................................................................25! 1.7.3.4. The MIA pathway....................................................................................26! 1.7.3.5. TIM22 complex.......................................................................................27! 1.8. Nucleic acid import................................................................................................29! 1.8.1. DNA import into mitochondria.......................................................................29! 1.8.2. RNA import into mitochondria.......................................................................29! 1.8.2.1. RNA import into protozoans mitochondria.............................................30! 1.8.2.2. RNA import into Plant mitochondria.......................................................33! 1.8.2.3. RNA import in Yeast mitochondria.........................................................35! 1.8.2.4. RNA import in mammals.........................................................................39! 2. Results..........................................................................................................................49! 2.1. Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase.................................................................................49! 2.1.1. Summary.........................................................................................................49! 2.1.2. Publication 1..................................................................................................52! 2.2. Human enolases facilitate tRK1-preKARS2 complex formation..........................53! 2.3. Mutation in PNPT1, which Encodes a Polyribonucleotide Nucleotidyltransferase, impairs RNA import into mitochondria and causes respiratory chain deficiency..56! 2.3.1. Summary.........................................................................................................56! 2.3.2. Publication 2..................................................................................................58! 2.4. PNPase overexpression increased RNA import into mitochondria.......................59! 2.5. PNPase down-regulation decreased RNA import into mitochondria....................61! 2.6. Identification of proteins interacting with PNPase................................................62! 3. Discussion....................................................................................................................59! 3.1. preKARS2 interacts with yeast tRK1 and its derivatives......................................59! 3.2. Human enolases facilitate tRK1-preKARS2 complex formation..........................60! 3.3. preKARS2 as a tRK1 carrier into human mitochondria........................................61! 3.4. Mitochondrial import of small truncated RNAs....................................................62! 3.5. PNPase translocate RNA into the mitochondrial matrix.......................................63! 3.6. PNPase mutation affect mitochondrial translation................................................64! TABLE OF CONTENTS 3.7. Divergent mechanisms for convergent goal..........................................................65! 4. Conclusions and perspectives....................................................................................73! 5. Materials and Methods...............................................................................................76! 5.1. Materials................................................................................................................76! 5.1.1. Bacterial strains...............................................................................................76! 5.1.2. Human cell lines.............................................................................................76! 5.1.3. Plasmids and oligonucleotides........................................................................77! 5.2. Methods.................................................................................................................78! 5.2.1. Electroporation of E. coli................................................................................78! 5.2.2. Plasmid DNA preparation...............................................................................78! 5.2.3. Recombinant protein purification...................................................................78! 5.2.4. Polymerase chain reaction (PCR)...................................................................79! 5.2.5. RNA synthesis................................................................................................80! 5.2.6. Radioactive labeling and aminoacylation.......................................................80! 5.2.7. Radioactive labeling of oligonucleotides probes............................................81! 5.2.8. Electrophoretic mobility shift assay (EMSA).................................................81! 5.2.9. Cell culture......................................................................................................82! 5.2.10. Transfection of human cell lines...................................................................83! 5.2.10.1. Transfection with T7 transcript..............................................................83! 5.2.10.2. Transient transfection with plasmid.......................................................84! 5.2.10.3. Transient protein downregulation..........................................................84! 5.2.11. Purification of mitochondria.........................................................................85! 5.2.12. In vitro import assay.....................................................................................86! 5.2.13. Northern hybridization..................................................................................86! 5.2.14. Immunoblotting.............................................................................................87! 5.2.15. Mitochondrial translation analysis in vivo....................................................88! 5.2.16. North-western blot........................................................................................88! 5.2.17. Cross linking immuno precipitation (CLIP).................................................89! 5.2.18. Mass Spectromery Analysis..........................................................................89 BIBLIOGRAPHY........................................................................................................................91 RÉSUMÉ DU THÈSE...............................................................................................................118 LIST OF FIGURES Figure 1: Mitochondrion.................................................................................................................2! Figure 2: Schematic of the mitochondrial electron transport chain................................................4! Figure 3: The human mitochondrial genome..................................................................................6! Figure 4: Mitochondrial DNA replication machinery ...................................................................8! Figure 5: Models of mitochondrial DNA replication...................................................................11! Figure 6: Schematic representation of the D-loop regulatory region...........................................13! Figure 7: Targeting and Sorting Signals of mitochondrial precursor proteins.............................21! Figure 8: The presequence pathway of protein import into mitochondria....................................23! Figure 9: Sorting and assembly machinery (SAM) of outer mitochondrial membrane...............26! Figure 10: The machinery for import and assembly (MIA) of preprotein into mitochondrial intermembrane space....................................................................................................................27! Figure 11: The carrier pathway of preprotein import into mitochondria......................................28! Figure 12: Overview of mitochondrial tRNA-import channels....................................................34! Figure 13: Lysine tRNAs in S. cereviseae....................................................................................36! Figure 14: Mitochondrial tRNA import in S. cerevisiae...............................................................37! Figure 15: Structure of Human 5S rRNA.....................................................................................42! Figure 16: Hypothetical mechanism of 5S rRNA intracellular traffic..........................................44! Figure 17: Predicted structures of the yeast tRNALys(CUU) and small synthetic RNAs...............46! Figure 18: Protein purification and electrophoretic mobility shift assay......................................53! Figure 19: Interaction between labeled tRK1 and preKARS2 in the absence or presence of human enolases.............................................................................................................................55! Figure 20: Implication of PNPase in mitochondrial RNA import in vivo....................................60! Figure 21: Implication of PNPase in mitochondrial RNA import in vivo....................................62! ACKNOWLEDGEMENTS First of all, with utmost pleasure, I express my heartfelt and profound gratitude to Dr. Ivan Tarassov, my PhD supervisor, whose guidance, unflinching support and valuable feedback made this work possible. Thank you Ivan, for giving me the opportunity to join your lab, for introducing me to the field of mitochondrial research, for administrative and financial support during my 4th year of PhD, for your kind scientific assistance and for the great moments I passed in your garden parties. I am highly indebted to Dr. Nina Entelis, my additional supervisor, for her excellent continuous support and guidance throughout my PhD. I am thankful to you Nina, for your kind advices and assistance, for your personal scientific interests in my work, and for your help in interpreting my experimental results on daily basis. I am extremely grateful to Prof. André Schneider, Dr. Anne Lombès, and Dr. Phillipe Giegé, for accepting to be the members of my thesis jury, for their precious time and efforts to evaluate my thesis. I am also thankful to Dr. Mikhail Vysokikh, a former postdoctoral fellow, who was my scientific guru during the last three years of my PhD. Mike you really were an invaluable help, and I want to thank you for all the scientific knowledge you taught me, for your brilliant assistance in every situation and for our close and fruitful cooperation. I wish you all the best for your bright future in Moscow. I would like to extend my gratitude to all other members of our “Mito” group, past and present, especially Dr Alexandre Smirnov “Sacha”, Anne-Marie Heckel, Yann Tonin, Dr Tom Schirtz, Dr Caroline Comte, and Ilya for creating a scientifically motivating environment, for all their help, support, suggestions and friendship. I also want to thank all our members of the UMR7156 for the great and very stimulating working atmosphere and for their kind help and discussions. I felt very happy among you during the past four years and I really enjoyed all our nice parties. I am grateful to Higher Education Commission (HEC) of Pakistan, SFERE (Société française d’exportation des resources éducatives), and Ivan Tarassov lab, for financing my study in France. Finally, I am thankful to all my family members who have stood alongside me all these years. Words are meaningless to what they have given me. It is, I believe their prayers and spiritual support, which were a constant source of strength and inspiration to me that despite, many constraints, I was able to complete my work. Gowher Ali ABBREVIATIONS ABBREVIATIONS ADP adenosine diphosphate ANT adenine nucleotide transporter ATAD3 ATPase family AAA domain-containing protein 3 ATP adenosine triphosphate BB breakage buffer BSA bovine serum albumin CLIP crosslinking combined to immunoprecipitation CSB conserved sequence block DEPC diethylpyrocarbonate DMEM dulbecco modified Eagle's medium DNA deoxyribonucleic acid Drp1 dynamin related protein 1 DTT dithiothreitol E.coli Escherichia coli EDTA ethylenediaminetetraacetic acid EMSA electrophoretic mobility shift assay Eno-2 enolase-2 FADH2 flavin adenine dinucleotide hydroquinone form FRET förster resonance energy transfer HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMG high mobility group HSP heavy strand promoter Hsp heat-shock protein ABBREVIATIONS IFs initiation factors IMS intermembrane space IPTG isopropyl ß-D-1-thiogalactopyranoside KDa kilo Dalton LB lysogeny broth LSP light strand promoter LSU large subunit Mfn mitofusin mtDNA mitochondrial DNA mtEFTu mitochondrial elongation factor Tu mtEFTs mitochondrial elongation factor Ts mtEFG mitochondrial elongation factor G mtRF mitochondrial release factor mtRPOL mitochondrial RNA polymerase mtSSB mitochondrial single stranded DNA binding protein MIA mitochondrial intermembrane space import and assembly MISS mitochondrial intermembrane space signal MMP mitochondrial processing peptidase MRPs mitochondrial ribosomal proteins MTS mitochondrial targeting sequence mRNA messanger RNA NADH nicotinamide adenine dinucleotide reduced OD optical density ABBREVIATIONS OPA1 optic atrophy 1 PAM presequence translocase associated motor PBS phosphate buffered saline PNPase polynucleotide phosphorylase POLG pitochondrial DNA polymerase ! PPR pentatricopeptide repeat RIC RNA import complex RITOLS RNA incorporation throughout lagging strand ROS reactive oxygen species rRNA ribosomal RNA PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction preMSK precursor of Saccharomyces cerevisiae mitochondrial lysyltRNA- synthetase preKARS2 precursor form of human mitochondrial lysyltRNA-synthetase SAM sorting and assembly machinery S.cerevisiae Saccharomyces cerevsisae SDS sodium dodecylsulfate SELEX systematic Evolution of Ligands by Exponential Enrichment siRNA small interferring RNA SSC saline sodium citrate buffer ssDNA single stranded DNA TAB tubulin antisense binding protein TAS termination associated sequence

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Université de Strasbourg. 2013. Ecole doctorale des Soutenue le 17 September 2013 devant le jury de thèse composé comme suit: Professor Andre
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