THÈSE DOCTEUR DE L'UNIVERSITE DE STRASBOURG GOWHER Ali

164 Pages·2014·6.2 MB·French

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