What Are Their Roles in Mitochondrial Protein Synthesis?
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Characterisation of human mtRF1 and C12orf65: What are their roles in mitochondrial protein synthesis? Aleksandra Pajak M.Res Thesis submitted to Newcastle University in candidature for the degree of Doctor of Philosophy Newcastle University Faculty of Medical Sciences Institute for Ageing and Health Mitochondrial Research Group January 2013 Abstract Mitochondria have their own protein synthesis machinery that synthesises the oxidative phosphorylation components encoded by their mtDNA. This translation process consists of four main phases: initiation, elongation, termination and ribosome recycling. Termination and its control have been the least investigated. Recently, however, the termination factor, mtRF1a, has been characterised as sufficient to release all the nascent proteins from the mitoribosome. Furthermore, bioinformatics has identified three additional members of this mitochondrial release factor family namely, mtRF1, C12orf65 and ICT1. The latter is now known to be incorporated into the mitoribosome but its exact function remains unclear. My project has therefore focussed on characterising the remaining two factors; mtRF1 and C12orf65, and investigating their possible involvement in mitochondrial protein synthesis. It has been demonstrated that protein synthesis is not perfect and bacterial ribosomes not infrequently stall during translation. This can result from limiting amounts of charged tRNAs, stable secondary structures, or truncated/degraded transcripts. Ribosome stalling has been shown to cause growth arrest. In order to prevent that and maintain high efficiency of mitochondrial protein synthesis such stalled complexes need to be rapidly recycled. Bacteria have developed at least three distinct mechanisms by which ribosomes can be rescued. Contrastingly, despite the presence of truncated mRNAs in mitochondria, no such quality control mechanisms have been identified in these organelles. This study investigates the potential role of mtRF1 and C12orf65 in quality control of protein synthesis in mitochondria. Both mtRF1 and C12orf65 demonstrate conservative motifs which would suggest their potential role in ribosome rescue. My findings indicate that the conserved motifs in mtRF1 are crucial to maintain normal cell metabolism and that its mutated forms negatively affect cell growth. Since these motifs are required for ribosome dependent peptidyl-tRNA hydrolysis, the data presented strongly imply that mtRF1 plays a crucial role in intra-organellar protein synthesis. 2 Acknowledgments I would like to express my sincere gratitude to my supervisors, Prof. Zofia Chrzanowska-Lightowlers and Prof. Robert Lightowlers for providing me with the opportunity to study mitochondrial protein synthesis as a member of their group. They have been a steady influence throughout my Ph.D. studies, their scientific expertise, advice, but also many anecdotes have been invaluable to me and their hard work have set an example for many years. I would also like to thank them for their kindness, patience and all the encouragement I needed during difficult times. This work would not have been possible without their constant support and motivation; it has been a real pleasure to curry out my research in this laboratory. Additionally, I have been privileged to meet and collaborate with many fantastic people, who also become good friends over last 4 years. I would like to thank Ricarda Richter for all the time spent with me on the bench, teaching and helping me. Also I thank Sven Dennerlein, who introduced me to the Northern Blotting techniques. All the people, who I worked with everyday and who have been my 'secodn', extended family, thank you Agata, Francesco, Alina, Tran, Marysia, Casey, Kyle, Martin and Abdulraheem for the sharing of knowledge (not only scientific) many discussions, maintaining a good atmosphere in the lab and a good laugh at times. Thanks to all the members of the Mitochondrial Research Group (MRG). I thank the Biotechnology and Biological Sciences Research Council (BBSRC), Centre for Brain Ageing and Vitality (CBAV) and Newcastle University for funding and providing excellent working facilities. I owe special thanks to Karolina Rygiel, Agata Rozanska and Justyna Colliander. Thank you for being patient listeners to my worries, for many stimulating conversations and for being there for me always. Without your everyday moral support and all the help, when I needed it, I would not been able to make it this far and complete this thesis. Finally, I would like to thank my parents and the rest of my family for their understanding, the support from the distance and love. 3 Statement of originality I declare that the data presented in this thesis is based solely work carried out by the author, unless stated otherwise. Moreover, this thesis has not been submitted before for any other degree or award. The contributions by others were acknowledged, where appropriate, Aleksandra Pajak 4 Table of contents Chapter 1. Introduction…………………………………………………………..18 1.1. Mitochondria- the importance and uniqueness .............................................18 1.1.1. Evolution of Mitochondria ..................................................................................18 1.1.2. General Structural Characteristics of mitochondria ............................................19 1.1.3. Mitochondrial Functions ....................................................................................20 1.1.4. The human mitochondrial genome, its maintenance and transcription................22 1.2. Mitochondrial protein synthesis in human organelles ..................................25 1.2.1. Characteristics of the mammalian mitochondrial ribosome .................................28 1.2.2. Initiation of mitochondrial protein synthesis ........................................................37 1.2.3. Elongation of mitochondrial translation ..............................................................39 1.2.4. Termination of mitochondrial translation ............................................................41 1.2.5. Ribosome recycling in mitochondria ..................................................................42 1.2.6. Human mitochondrial release factor family ........................................................43 1.3. Overview of gene expression quality control ................................................46 1.1.1. Prokaryotic mRNA surveillance pathways..........................................................47 1.1.1.1. Trans translation ...........................................................................................47 1.1.1.2. Other ribosome-rescue pathway ...................................................................49 1.1.2. Eukaryotic mRNA surveillance pathways ...........................................................51 1.1.2.1. Nonsense-mediated mRNA decay (NMD) .....................................................51 1.1.2.2. Nonstop mRNA decay (NSD)........................................................................52 1.1.2.3. No-go decay (NGD) ......................................................................................53 1.1.3. Release factors and protein quality control ........................................................54 1.4. The aims of this study ....................................................................................55 Chapter 2. Materials and Methods………………………………………………59 2.1. Plasmid manipulations and use of DNA oligonucleotides. ...........................59 2.2. Cell culture ......................................................................................................61 2.2.1. Cell culture maintenance, storage and microscopy ............................................61 2.2.2. Cell counting .....................................................................................................61 2.2.3. Mycoplasma testing ..........................................................................................62 2.2.4. Forward and reverse siRNA transfection of HeLa and HEK293T cell lines. ........62 2.2.5. Stable Transfection of HEK293-Flp-InTMT-RexTM cells. ...................................62 2.3. Bacterial strains and general bacterial culture ..............................................63 2.3.1. Transformation of bacterial strains with plasmids. ..............................................63 2.3.2. Colony screening ..............................................................................................63 2.3.3. Plasmid DNA purification ...................................................................................64 2.4. DNA manipulation ...........................................................................................64 2.4.1. Polymerase chain reaction (PCR)......................................................................64 2.4.2. Purification of PCR products .............................................................................65 2.4.3. Restriction enzyme digestion .............................................................................66 2.4.4. Dephosphorylation of linearised vectors ............................................................66 2.4.5. Phenol/chloroform extraction and precipitation of DNA ......................................66 2.4.6. Ligation .............................................................................................................66 2.4.7. Electrophoresis .................................................................................................66 2.4.8. Site Directed Mutagenesis.................................................................................67