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Identification and characterisation of novel factors involved in the nonsense-mediated mRNA decay (NMD) pathway Angela Casadio Thesis presented for the degree of Doctor of Philosophy The University of Edinburgh 2016 Declaration I declare that this thesis is of my own composition. The work presented in this thesis is my own, unless otherwise stated, and has not been presented for any other degree or professional qualification. Angela Casadio ii Acknowledgements First and foremost, I would like to thank my supervisor Javier for making this possible. I have really enjoyed working on a field I knew little about when I started my project, I’ve learnt a lot in the past four years and I will treasure the experience! A huge thank you goes to Dasa for all the help and good advice she gave me during the course of my PhD. I will always be in your debt for being so patient with my last minute (or even last second) deadlines! Thanks, I really really appreciated it! Thanks to Nele for being an inexhaustible source of knowledge and for bearing with me when I was asking thousands of questions. You’ve made my life a lot easier and you’ve taught me a lot! To all past and present people in the lab, Elena, Fiona, Julie, Laurent, Magda, Marianne, Noemi, Ross, Sara, thanks for all the good times and the giggling! You made my time in the lab more enjoyable, and I will always treasure some of our conversations (how could I forget the floppy disc...). A special thank you to my “lunch pals”, Giulia, Raffaele, Sara and Silvia, for making my breaks enjoyable and for all the laughs. Our lunches were a pleasant escape to the day-to-day routine in the lab. Thanks to my tissue culture partner, Christine, who shared with me the pain of many hours and the complaints for the state of tissue cultures (and sorry if I complained too much). I was always relieved to come to TC and find a friendly face! I would also like to thank all the people that helped me one way or another by providing advice and reagents. In particular I’d like to thank Prof. Oliver Mühlemann for the HeLa β-globin cells and Andy Finch for the gift of the siRNAs and for good advice. Thanks to Richard, my second supervisor, Ian and Greg for the usuful suggestions and feedback. iii Thanks to Silvia, Bob, Richard M and Ian J for their supervision during the 6-week rotations. With you I have learnt the basic techniques that helped me progress during my PhD project. Thanks for broadening my knowledge in various fields of biology. I would like to thank all the people in the technical service, in particular Sean, Stephen and Stewart, for making my life easier and helping me when the robot got stuck again and again... Thanks to Matt from the microscopy service for helping to set up the microscope almost every single time! Also thanks to Jimi and Graeme for helping me with the processing and analyses of the mass spec and microarray data. Grazie a Anne, Amy e Erica, per farmi sentire il calore di una famiglia anche lontano da casa. Un grazie speciale alla mia famiglia per il costante supporto e l’affetto. Grazie per sopportare la lontananza, ma essere sempre presenti. Questo mio traguardo è stato possibile solo grazie a voi. Vi voglio bene! Infine, last but not least, grazie di cuore al mio ragazzo Mattias, senza di te non sarebbe stato possibile! Grazie per aver sopportato i miei sbalzi di umore in quest’ultimo periodo e per avermi amata! Grazie grazie grazie! Ti amo. iv Abstract Nonsense mediated mRNA decay (NMD) is a surveillance mechanism that targets transcripts containing premature stop codons (PTCs) for degradation, and that also regulates up to 10% of the whole transcriptome. During the course of my PhD I set out to identify novel NMD factors by performing a genome-wide RNA interference (RNAi) screen in a transgenic strain of Caenorhabditis elegans carrying an NMD reporter. I identified five novel proteins that are putative NMD factors in worms: NGP-1, NPP-20, AEX-6, PBS-2 and NOAH-2. Knock-down of these proteins led to severe developmental defects: worms were either arrested during various larval stages or died prematurely. The only exception was AEX-6, the knockdown of which led to a milder phenotype. Homology analysis of the novel C. elegans NMD factors showed that these proteins are conserved in human, with the exception of NOAH-2, which only has a homologue in Drosophila melanogaster, NOMPA. By performing an NMD assay in human cells, I demonstrated that GNL2 (NGP-1) and SEC13 (NPP-20) are functionally conserved NMD factors in human. Analysis of the consequences of depletion of GNL2, SEC13, UPF1 or UPF2 on the transcriptome of HeLa cells revealed that these four proteins co-regulate a subset of endogenous NMD targets, whilst also independently regulating the expression of other sets of transcripts. The findings presented in this thesis further our knowledge of the biology of NMD in both nematodes and humans. They demonstrate the existence of further regulators of this surveillance pathway, and add a layer of complexity to this fine-tuned biological process. v Lay summary DNA contains the genetic information that cells need to grow and to properly function. To use this information, DNA is transcribed to messenger RNA (mRNA) that is then translated into proteins. This process is crucial for the survival of the cells. Several mechanisms have therefore been developed by organisms during the course of evolution to check that the genetic information is correctly interpreted. One of these mechanisms is called nonsense-mediated mRNA decay (NMD). Its role is to eliminate mRNAs that would result in proteins shorter than normal ones, which may interfere with the correct functioning of cells. NMD has been associated with several diseases, such as β- thalassemia, muscular dystrophy and intellectual disorders. Functioning NMD is important for proper development of mammals, as the NMD pathway is responsible for regulating up to10% of all mRNAs. This quality control process has been studied for over twenty years and we already know several of its players. However, not all steps of the NMD pathway are clear, so more effectors could still await discovery. The aim of my PhD project was to find new factors involved in NMD and to try to understand their role in NMD. I found five putative NMD effectors in the nematode worm Caenorhabditis elegans (NGP-1, NPP-20, AEX-6, PBS-2, and NOAH-2). Two of the newly identified factors are also functionally conserved in humans (GNL2/NGP-1, and SEC13/NPP-20). I have also shown that GNL2 and SEC13 collaborate with known NMD factors to regulate physiological mRNAs. This work has increased our understanding of how NMD is regulated, and this deeper understanding will hopefully aid the scientific community in the study of how this process regulates normal development and disease. vi Table of contents Declaration ii Acknowledgements iii Abstract v Lay summary vi Table of contents vii List of figures xii List of tables xv List of abbreviations xvi Chapter 1: Introduction 1 1.1 When an mRNA has been translated correctly: mechanisms of normal translation termination 3 1.2 When the life an mRNA is over: cytoplasmic mRNA degradation 5 1.3 When the ribosomes stop elongating: no-go decay (NGD) 7 1.4 When messenger RNAs lack the stop codon: non-stop decay (NSD) 8 1.5 When ribosomes encounter premature stop codons: nonsense-mediated decay (NMD) 8 1.5.1 The NMD factors are conserved throughout evolution in eukaryotes 9 1.5.2 Transcripts bearing a premature stop codon: mechanism of NMD 12 1.5.3 Alternative mechanism of NMD in mammalian cells 21 1.5.4 Physiological importance of NMD 22 1.5.4.1 The role of NMD in development 22 vii 1.5.4.2 The role of NMD in disease 24 1.6 A relative of NMD: staufen-mediated mRNA decay (SMD) 26 1.6.1 Functions of staufen 28 1.7 Project outline 29 Chapter 2: Materials and Methods 31 2.1 Materials 31 2.1.1 Solutions 31 2.1.2 C. elegans strain 33 2.1.3 Cell lines 33 2.1.4 Primers 34 2.1.5 Expression vectors 35 2.1.6 List of materials 36 2.2 Methods 38 2.2.1 Maintenance of C. elegans 38 2.2.2 Synchronisation of population of worms 38 2.2.3 The screen 39 2.2.3.1 Description of the RNAi library 39 2.2.3.2 Description of screen strategy 39 2.2.3.3 Visualisation of GFP-expressing worms 40 2.2.3.4 Determination of clones identity 40 2.2.4 Imaging of worms 41 2.2.4.1 Preparation of slides 41 2.2.4.2 Acquisition of images 43 2.2.5 Maintenance of cell lines 43 2.2.6 siRNA treatment 43 2.2.7 Actinomycin D treatment 44 2.2.8 RNA extraction 44 2.2.8.1 Extraction of RNA from C.