HUMAN RIBOSOME BIOGENESIS AND THE REGULATION OF THE TUMOUR SUPPRESSOR p53 Andria Pelava Submitted for Doctor of Philosophy Final submission: December 2016 Institute of Cell and Molecular Biosciences Faculty of Medical Sciences Newcastle University ii Abstract Ribosome production is an energetically expensive and, therefore, highly regulated process. Defects in ribosome biogenesis lead to genetic diseases called Ribosomopathies, such as Dyskeratosis Congenita (DC), and mutations in ribosomal proteins and ribosome biogenesis factors are linked to multiple types of cancer. During ribosome biogenesis, the ribosomal RNAs (rRNAs) are processed and modified, and defects in ribosome biogenesis lead to the activation of p53. This project aimed to investigate the functions of Dyskerin, mutated in X-linked DC, in human ribosome biogenesis and p53 regulation, and to explore the link between ribosome production and p53 homeostasis. Dyskerin is an rRNA pseudouridine synthase and required for telomere maintenance. There is some debate as to whether DC is the result of telomere maintenance or ribosome biogenesis defects. It is shown here that human Dyskerin is required for the production of both LSU and SSU, and knockdown of Dyskerin leads to p53 activation via inhibition of MDM2 via the 5S RNP, an LSU assembly intermediate which accumulates after ribosome biogenesis defects. My data indicate that p53 activation, due to defects in ribosome biogenesis, may contribute to the clinical symptoms seen in patients suffering with DC. In addition, it is shown that defects in early or late stages of SSU or LSU biogenesis, result in activation of p53 via the 5S RNP-MDM2 pathway, and that p53 is induced in less than 12 hours after ribosome biogenesis defects. SSU defects do not cause any obvious defects in LSU production, but they result in inhibition of export of the pre-LSU in the cytoplasm, suggesting that p53 activation after SSU defects is probably due to defects in pre-LSU export. Finally, there are evidence that RPS27a or RPL40, two ribosomal proteins produced as ubiquitin-fusion precursors in the cell, might be novel regulators of the 5S RNP-MDM2 pathway. In conclusion, this work shows the importance of ribosome biogenesis in the regulation of p53 for the development of Ribosomopathies and cancer. iii iv Declaration I certify that the work presented here is my own, unless when acknowledged. No part of this work has been previously submitted for other qualifications at any university. Acknowledgements I would like to thank my supervisor, Dr Nick Watkins, for all his help and support throughout my PhD, a character-building process from which I have learnt a lot as a scientist and as a person. I would also like to thank Dr Claudia Schneider for her advice and help over the past few years, and everyone in the Watkins/Schneider lab for putting up with me! Special thanks to Dr Loren Gibson for her help during experimental procedures and her support since the beginning. Thank you to Dr Katherine Sloan and Dr Christopher Hoyland for their precious help at the beginning of my PhD, to Dr Neil Perkins for providing some of the human cell lines used and to my panel assessors, Dr Elizabeth Veal and Dr Viktor Korolchuk, for their help and advice during my PhD. Furthermore, I would like to thank my friends Dr Claire Whitworth and Kerrie Brusby for the coffee breaks during stressful days, Ellouise for her valuable advice and inspiration, and Charis for all her support from far away. A huge thank you to Rozi and Miriam for their understanding and constant support throughout, and to all of my Capoeira family, especially to Will, Patrick, Rob and Glenn. Special thanks to Jimi, who has supported me like a second father while I was away from home. I would like to thank my “kouklitsa” Eleni; you have been my rock since the beginning and I could not have done this without you. Last but not least, I would like to thank my sister, Christia, my brother, Stylianos and my parents, Nikos and Eftychia, for being the best parents I could have asked for. I could not have done any of this without your support and your love. Thank you for everything you have taught me and for being there no matter what. This work was funded by the BBSRC-DTP and the Diamond-Blackfan Anaemia Foundation, U.K. v vi Table of Contents Abstract .................................................................................................................... iii Declaration ............................................................................................................... v Acknowledgements .................................................................................................. v Table of Contents .................................................................................................... vii List of Figures ........................................................................................................... xi List of Tables .......................................................................................................... xiv List of Abbreviations ................................................................................................ xv 1. Chapter One. Introduction .................................................................................. 1 1.1. The eukaryotic ribosome ................................................................................ 1 1.2. Ribosome Biogenesis ..................................................................................... 1 1.2.1. The nucleolus .......................................................................................... 2 1.2.2. rRNA transcription ................................................................................... 3 1.2.3. rRNA processing ...................................................................................... 4 1.2.4. Ribosomal protein production ................................................................ 10 1.2.5. The 5S RNP........................................................................................... 11 1.2.6. Recruitment of the 5S RNP in the large ribosomal subunit .................... 12 1.2.7. Small and large ribosomal subunit assembly and proofreading ............. 13 1.2.8. Nuclear export ....................................................................................... 14 1.3. Ubiquitin-ribosomal protein genes ................................................................ 16 1.3.1. The importance of ubiquitin in the cell ................................................... 16 1.3.2. RPRS27a- and RPL40-ubiquitin fusion ribosomal proteins ................... 19 1.4. Modifications of ribosomal RNA ................................................................... 20 1.4.1. Pseudouridylation and methylation of rRNA .......................................... 20 1.4.2. The Box C/D snoRNP ............................................................................ 21 1.4.3. The H/ACA snoRNP .............................................................................. 23 1.4.4. Other functions of the H/ACA RNP ........................................................ 26 1.4.5. The pseudouridine synthase Dyskerin ................................................... 28 1.5. Ribosomopathies ......................................................................................... 29 1.5.1. Introduction ............................................................................................ 29 1.5.2. Diamond-Blackfan Anaemia .................................................................. 31 1.5.3. 5q- syndrome ......................................................................................... 32 1.5.4. Treacher-Collins syndrome .................................................................... 32 vii 1.5.5. Dyskeratosis Congenita ........................................................................ 33 1.5.6. Ribosomes, Ribosomopathies and cancer ............................................ 35 1.6. The tumour suppressor p53 ......................................................................... 36 1.6.1. p53 ........................................................................................................ 36 1.6.2. p53 regulation and activation ................................................................ 37 1.6.3. Cell cycle regulation by p53 .................................................................. 39 1.6.4. p53 function in apoptosis....................................................................... 40 1.6.5. p53 function in DNA damage repair ...................................................... 42 1.6.6. The 5S RNP-MDM2 pathway for p53 regulation ................................... 43 1.7. Aims and Objectives .................................................................................... 45 2. Chapter Two. Materials and Methods ............................................................... 48 2.1 DNA methods .............................................................................................. 48 2.1.1 Construct introduction ........................................................................... 48 2.1.2 Polymerase Chain Reaction (PCR) for cloning ..................................... 48 2.1.3 DNA visualization and extractions ......................................................... 49 2.1.4 Ligation in pJET1.2 vector ..................................................................... 49 2.1.5 Transformations in Escherichia coli and DNA extractions ....................