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Development of a New Screening System for the Identification of RNF43-Related Genes and Characterisation of Other PA-RING Family Members

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Development of a New Screening System for the Identification of RNF43-Related Genes and Characterisation of Other PA-RING Family Members Development of a new screening system for the identification of RNF43-related genes and characterisation of other PA-RING family members Alessandra Merenda University of Cambridge Department of Genetics This dissertation is submitted for the degree of Doctor of Philosophy Darwin College April 2017 ii | P a g e This dissertation is dedicated to my exemplary grandfather. iv | P a g e Declaration I hereby declare that, except where specific reference is made to the work of others, the contents of this dissertation are original and they are the result of my own work. This dissertation contains nothing which is the outcome of work done in collaboration with others, except as specified in the text and under “External contributions”. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution. This dissertation does not exceed the prescribed word limit of 60,000 words excluding bibliography, figures and appendices. Alessandra Merenda April 2017 v | P a g e External contributions The details about external contributions to the work presented in this thesis are as follows: Dr. Ingrid Jordens (UMC) and Dr. Cara Jamieson (UMC) performed the pulse chase experiments with SNAP-Frizzled5 (Figure 5.2 and Figure 5.3). Dr. Jihoon Kim (University of Cambridge) performed FZD5 western blot and FACS analysis, ubiquitination assay and co-immunoprecipitation (Figures 5.4-5.7). vi | P a g e vii | P a g e Acknowledgments As this intense journey comes to an end, many are the people I feel like thanking for making it worth undertaking. I would firstly like to thank Bon-Kyoung for his great supervision and guidance, his support and encouraging words whenever needed during these years and, of course thank you to all the current and past Koo lab members. In particular, thanks to Jihoon for intensively collaborating with me on my main project. In addition, I appreciate the support received from the Stem Cell Institute core facilities, especially the Tissue Culture, Flow Cytometry, Histology and Animal facilities. A special thank goes to Chris Hindley and Luigi Aloia for patiently reading and proofreading this thesis and giving me plenty of insightful feedbacks. I am also very grateful to Meritxell Huch (Gurdon Institute) and Joo-Hyeon Lee (SCI) for their valuable advice and feedbacks on my progress, and to everyone in their labs. In particular, thanks Mastrogiova’ for being such an amazing PhD buddy and friend! Moreover, I would like to thank my advisors Marisa Segal and Alfonso Martinez-Arias (Department of Genetics) for providing feedback on my first and second year reports and following my progress since I joined the Department. I would also like to thank the Seventh Framework Program, Marie Curie Actions and the European Union for generous funding of my project and Madelon Maurice (University Medical Centre Utrecht) for mentoring me within the Wntsapp network. In her lab, I am particularly grateful to Ingrid for being such a great Wntsapp project manager and for collaborating with me together with Cara, and to Nicola for his friendship and all the scientific and non-scientific discussions. viii | P a g e Then, many thanks also to all the other Wntsapp PIs and fellows for their great inputs and the many fun times shared together. This PhD experience wouldn’t have been the same without you! …And it would not have been the same either without my friends, far or near, old and new. Thanks for bringing a smile on my face as well as putting up with me! Finally, the biggest thank you I for sure owe it to my family. Grazie per essermi stati così vicini anche da lontano durante questo intenso cammino di formazione, ma soprattutto grazie per l’inestimabile bagaglio con cui avete fatto in modo che partissi. ix | P a g e x | P a g e Title: Development of a new screening system for the identification of RNF43- related genes and characterisation of other PA-RING family members. Candidate: Alessandra Merenda Abstract The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the xi | P a g e investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation. xii | P a g e Table of contents Acknowledgments .......................................................................................................................... viii Abstract ............................................................................................................................................. xi Table of contents ............................................................................................................................ xiii List of figures ....................................................................................................................................xvi List of tables ..................................................................................................................................... xix Abbreviations....................................................................................................................................xx Chapter 1 - Introduction .................................................................................................................... 1 1.1.1 Architecture of the adult small intestine ......................................................................... 1 1.1.2 The stem cell niche and signalling pathways ................................................................... 4 1.2 Modelling the small intestine in vitro ......................................................................................... 7 1.2.1 Establishment of intestinal organoid culture .................................................................. 7 1.2.2 Applications of organoid technology ............................................................................... 9 1.2.3 Genetic manipulation of organoids ............................................................................... 10 1.3 CRISPR/Cas9 technology for genome editing ........................................................................... 12 1.3.1 CRISPR/Cas9 for knockout and precise gene editing .................................................... 12 1.4 WNT signalling pathways .......................................................................................................... 15 1.4.1 The “canonical” WNT/β-catenin pathway ..................................................................... 17 1.4.2 β-catenin independent WNT signalling pathways ........................................................ 18 1.5 RNF43 & ZNRF3, novel key players in WNT pathway regulation ............................................. 20 1.5.1 Rnf43 & Znrf3 are important tumour suppressors in the small intestine .................... 20 1.5.2 RNF43 and R-spondin are often mutated in cancer ...................................................... 24 1.5.3 Role of Dishevelled in the mechanism of action of RNF43 ........................................... 25 1.6 The PA-RING family of E3 ubiquitin Ligases.............................................................................. 27 1.6.1 The Goliath-related protein family ...............................................................................
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