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This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Uncovering non-canonical functions for the E3 ubiquitin ligase UBR2 in the mouse Eleanor Siân Raymond Presented for the degree of Doctor of Philosophy Genetics and Molecular Medicine The University of Edinburgh September 2019 Minor corrections accepted February 2020 0 Declaration I declare that the work presented within this thesis is my own, unless explicitly stated. The work has not been submitted for any other degree or professional qualification. Eleanor Siân Raymond 16th September 2019 1 2 Acknowledgements This thesis was a labour of love – and sometimes frustration – and it would not have been possible without the innumerable people who supported me, who deserve enormous gratitude and respect. Firstly, and most importantly, Ian Adams. He was a constant source of inspiration and motivation, and always had confidence in my ideas. Without him, this thesis would never have been conceived, and his constant support allowed me the freedom to take his idea and run with it. Members of the Adams Family, both past and present, were all largely responsible for my day- to-day sanity. James Crichton and Karen Dobie, in particular, were always available for advice, complaints and emotional support. Simon Brown, Veronica Duffy, Jennifer Lawson, Issy MacGregor and Apiwat Moolnangdeaw provided chats and snacks whenever required. Chris Playfoot, Marie MacLennan and Soña Relovska gave me the grounding in my project from the beginning. And our dear friend David Read, with his Spotify Premium and sunshine breaks, who saw the fun in every day in the lab. Thank you to every member of the Pub Club, whose empathy, PhD memes and pub quiz knowledge were vital in maintaining a sense of balance that we all sorely needed as we went through this experience together. To Frank, Hollie, those in the Constellation and everyone else – you allowed me an escape from the science, and you became a community in which I was incredibly included and supported throughout anything. And finally, my family – those who have never faltered in their confidence in my abilities, even when my own confidence was lacking. My parents, Diane and David Raymond, who instilled a great sense of curiosity in me from a young age. For mathematical advice, Daniel Cunnah. For IT and tech. support, Sean Casey. And for snacks and cheerleading, Charlotte Cunnah. Thank you. 3 4 “Opening your eyes is all that is needed. Look with your eyes. Hear with your ears. Taste with your mouth. Smell with your nose. Feel with your skin. Then comes the thinking, afterward, and in that way: knowing the truth.” – George R. R. Martin This work, and the research contained within it, are dedicated to Sean Casey. You kept me going, every morning and every night. The final days of this process felt almost insurmountable without the support of Kate, Charly, Daniel, Sophie and so many more, who gave me a roof over my head and a place to work. 5 6 Abstract Ubiquitination is an essential cellular mechanism in which the small polypeptide ubiquitin is attached to proteins, so regulating protein function or targeting them for degradation via the proteasome. The transfer of ubiquitin to specific target proteins is regulated by the highly diverse group of E3 ubiquitin ligases. These require two key domains: a substrate recognition site, and a ubiquitin carrier E2 protein binding site, which allow close proximity of the two, so ubiquitin transfer can occur. One family of E3 ubiquitin ligases are the UBR proteins, which confer substrate specificity through the N-end Rule pathway – in which they, canonically, bind either Type I (basic, e.g. Arginine) or Type II (bulky hydrophobic, e.g. Phenylalanine) amino acids on the N-terminus of the substrate. There are seven known UBR proteins, and UBR2 is one of four in this family that are thought to have overlapping roles in the N-end Rule pathway. Published murine phenotypes in a Ubr2-/- mouse include male infertility and female- specific embryonic lethality. The tissue-specificity of the Ubr2-/- phenotype has been attributed to tissue-specific expression of UBR proteins allowing critical functions of the N- end rule pathway to become detectable in specific cell types. These phenotypes also appear to overlap that of a Tex19.1-/- mouse, which has undetectable expression of the UBR2- interacting protein TEX19.1. This is specifically expressed in hypomethylated tissues: the testes, ovaries, placenta, and in a mouse embryonic stem cell model. In addition to the male infertility observed in both mouse models, Tex19.1-/- phenotypes include placental defects associated with intrauterine growth restriction, and cohesin misregulation during female meiosis, in the oocyte. There is some evidence that UBR2’s interaction with TEX19.1 regulates its Type II N-end Rule activity, and so I hypothesized that UBR2 also has roles in the other phenotypes of the Tex19.1-/- mouse, and that these act through the N-end Rule, which has thus far not been directly shown in any of the phenotypes present. Firstly, I aimed to study a potential role for UBR2 during female meiosis. In oocytes, cohesin molecules hold homologous chromosomes in a tightly aligned conformation. This must be 7 maintained for long periods of time, between meiotic prophase in the embryonic ovary and resumption of meiosis prior to ovulation during adulthood. This maintains chromosome stability and prevents missegregation during the first meiotic division, leading to aneuploidy and reduced fertility. A longer arrest period, i.e. in oocytes isolated from older females, is associated with loss of cohesin and increased chance of aneuploidy. In absence of UBR2 in oocytes from young females, less cohesin is present on the chromosome arms at the first meiotic metaphase and homologs are less tightly held together, so implying a role for protection of chromosome cohesion over time. Next, in accordance with previous observations in absence of UBR2, I identified an embryonic lethality present in our Ubr2-/- mice, which is associated with intrauterine growth restriction and placental defects. Although this is superficially similar to the Tex19.1-/- placental phenotype, there also appear to be TEX19.1-independent functions of UBR2 in this tissue. Finally, I aimed to ascertain the mechanism by which UBR2 plays a role in any of these phenotypes. As UBR2 is thought to confer particular specificity towards Type II N-end Rule substrates, and TEX19.1 is thought to regulate this role of UBR2, I used CRISPR-Cas9 technology to generate an Ubr2T2/T2 mouse model, which has undetectable Type II substrate binding. There are no gross phenotypes in any of the tissues identified using the Ubr2-/- null mouse; both placental growth and meiotic cohesin appear to be normally regulated, and so these functions of UBR2 do not appear to be acting through the N-end Rule pathway. Additionally, the well-established role for UBR2 during spermatogenesis does not appear to act through Type II N-end Rule substrate binding, as has been assumed. Instead, I conclude that, although UBR2 is an important part of the N-end Rule pathway in other contexts, it has key non-canonical functions in both male and female fertility, and in placental development, which cannot be compensated for by other UBR proteins. 8 Lay Abstract Proteins are the functional products of genes, and they act in cells to carry out the processes required for cells to survive. In order to keep cells or tissues functioning optimally, individual protein levels need to be tightly controlled, and so an efficient recycling process exists in order to “tag” proteins that need to be turned over – this is called ubiquitination. A small molecule, called ubiquitin, is transferred to a target protein by an ubiquitin ligase enzyme and this “tags” the protein, directing it towards a recycling centre called the proteasome. There are over 10,000 proteins in any given cell, and so target specificity is very important in order to recycle the correct proteins and ensure cell survival. One ubiquitin ligase, called UBR2, uses a process called the N-end Rule to specifically bind its target proteins. There are four UBR enzymes that are all thought to have overlapping roles in the N-end Rule pathway, but in a Ubr2 mutant mouse, in which UBR2 is not present to recycle its target proteins, there are a specific subset of tissues affected. This implies that UBR2 has unique functions in these tissues that cannot be covered by other UBR enzymes. Of these unique functions, the best studied prior to this thesis is its role in male fertility. I hypothesized that UBR2 is also important for female fertility and that both of these roles are regulated by UBR2’s N-end Rule activity, which has thus far not been directly shown. Firstly, I aimed to characterise poorly understood roles of UBR2 in female fertility. I have shown that UBR2 appears to regulate chromosome cohesion during egg (oocyte) production. All oocytes are present in the ovary during embryogenesis, and do not complete their development until immediately prior to ovulation, when pairs of chromosomes are divided in half.

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