OCCURRENCE & FUNCTION OF CELLULAR 2A SEQUENCES Claire Roulston A Thesis Submitted for the Degree of PhD at the University of St Andrews 2015 Full metadata for this item is available in St Andrews Research Repository at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/7062 This item is protected by original copyright This item is licensed under a Creative Commons Licence Occurrence & Function of Cellular 2A Sequences Claire Roulston This thesis is submitted in partial fulfilment for the degree of PhD at the University of St Andrews 2015 1. Candidate’s declarations: I, Claire Roulston, hereby certify that this thesis, which is approximately 60000 words in length, has been written by me, and that it is the record of work carried out by me, or principally by myself in collaboration with others as acknowledged, and that it has not been submitted in any previous application for a higher degree. I was admitted as a research student in September 2010 and as a candidate for the degree of Doctor of Philosophy in Molecular Biology in September 2011; the higher study for which this is a record was carried out in the University of St Andrews between 2011 and 2015. Date …… Signature of Candidate ……… 2. Supervisor’s declaration: I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of Doctor of Philosophy in Molecular Biology in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date …… Signature of Supervisor ……… 3. Permission for publication: In submitting this thesis to the University of St Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that my thesis will be electronically accessible for personal or research use unless exempt by award of an embargo as requested below, and that the library has the right to migrate my thesis into new electronic forms as required to ensure continued access to the thesis. I have obtained any third-party copyright permissions that may be required in order to allow such access and migration, or have requested the appropriate embargo below. The following is an agreed request by candidate and supervisor regarding the publication of this thesis: PRINTED COPY a) Embargo on all or part of print copy for a period of one year on the following ground(s): Publication would preclude future publication Supporting statement for printed embargo request: I intend to publish papers using the material presented in Chapters 3-8. ELECTRONIC COPY a) Embargo on all or part of electronic copy for a period of one year on the following ground(s): Publication would preclude future publication Supporting statement for electronic embargo request: I intend to publish papers using the material presented in Chapters 3-8. Date …… Signature of Candidate …… Signature of Supervisor ……… i ‘It is best to prove things by actual experiment; then you KNOW; whereas if you depend on guessing and supposing and conjecturing, you never get educated. Some things you CAN'T find out; but you will never know you can't by guessing and supposing: no, you have to be patient and go on experimenting until you find out that you can't find out. And it is delightful to have it that way, it makes the world so interesting.’ Eve's Diary – Mark Twain, 1905 ii Dedication This thesis is dedicated to the memory of George Rae, my honorary Grandfather, for literally showing me how to appreciate the ground beneath my feet and the wonders that can be encased in a rock-pebble. For the fossils, the good advice, and the days out in the beat-up old green Volvo. Without you, I would not be a Scientist, with love always, Claire. Acknowledgements First and foremost I would like to take this opportunity to thank my supervisor Martin Ryan for initially granting me asylum when I was a refugee doctoral student without a lab, and then for providing unceasing support and patiently sharing his expertise over the last three years. I’d like to thank all the members of the Ryan lab, both past and present, for all your help, encouragement and laughter. Extra special thanks are due to Dr. Garry Luke for teaching me the basics of cloning technique, John Nicolson for microscopy, and to Dr. Ekaterina Minskaia for just about everything else laboratory-based. Also to Fiona Tulloch, Ashley Pearson and Séan Wilson for all the office banter. I’ll miss you all. Thank you. I’d like to thank all the people round and about the BMSRC building who helped out with their expertise, spare reagents, free cake and general advice and support. Special thanks to Dr. Catherine Botting for mass spec analysis, and to Dr. Jens Tilsner for undertaking the plant infections. Thank you also to Dr. Lars Brunner at SAMS for gifting me the sea-urchin eggs. To all my friends who supported me, you know who you are, and thank you. Special thanks to Derek Simpson for all the impromptu outward bound courses. To Kay and Jennifer Drysdale, for merely being yourselves, and for loaning out Bluebell. To all my shipmates at the JST, particularly Iain, Sean, Steve, Mo and Graham for all the adventures and for teaching me to counter adversity with grace and humour, and to never ever give up, even come hell and high water. I’ve seen you sail through it all with smiles on your faces and long may you continue to do so. To my little ships, Swallow and Iolar, and our crewmates: I wish you fair winds and calm waters and a safe harbour at voyage end. Lastly, but very certainly not least, thanks to my family, particularly my parents, for all their love and support and belief in me (and taxi rides home!), and to my granddad, one hundred years young and full of life. iii iv Abstract This thesis describes experiments investigating the translational recoding activities and the novel dual signalling properties of eukaryotic ribosome skipping 2A sequences. Over twenty years ago, the 19 amino acid 2A region of a Picornavirus; namely, Foot-and-Mouth Disease Virus (FMDV) polypeptide was shown to possess apparent “self-cleaving” abilities, cutting at its own C-terminus during translation (Ryan et al., 1991). Active FMDV 2A-like sequences were subsequently found in a number of related viruses (Luke et al., 2008), with several now utilised as essential biotechnology multi-gene transfer tools (Luke et al., 2010b). Then, in 2006, eukaryotic 2A-like sequences were identified from trypanosome non-LTR sequences. These were found to be functional in vitro (Heras et al., 2006). I have been able to identify over 400 putative eukaryotic 2A-like sequences through searching the freely available online proteomic and genomic databases. Data is presented to show that these 2As were encoded in frame with non-LTRs, or metabolic, or immune function genes, from a wide range of eukaryotic organisms; but I could not discern any obvious phylogenetic distribution for 2A. I have discovered that the majority of eukaryotic 2A sequences tested can mediate ribosome skipping in vitro. Modelling in silico indicated that active 2A-like sequences possessed the propensity to form a central alpha-helical region, whereas the models suggested that inactive 2A-like sequences would be essentially unstructured. I also report that some of these eukaryotic 2A peptides constitute a novel form of dual protein targeting as they play a dual role as exocytic pathway signal peptides mediating extracellular protein trafficking. I have shown that this protein trafficking ability is evolutionarily conserved, with an echinoderm sequence able to direct protein targeting in both plant and mammalian cells. I therefore propose that these novel eukaryotic 2A sequences could potentially become extremely valuable in biotechnological engineering. v Table of Contents Abstract ............................................................................................................................................. v Table of Contents ............................................................................................................................ vi List of Figures ................................................................................................................................ xiii List of Tables ................................................................................................................................. xvi List of Abbreviations ................................................................................................................... xvii Chapter 1. Discovery of 2A-Like Sequences .................................................................................. 1 1.1 Viruses ..................................................................................................................................... 1 1.2 Positive Sense Single-Strand RNA Viruses – Translational Tricks ......................................... 2 1.3 Ribosomes ................................................................................................................................ 7 1.4 Ribosome Exit Tunnel ............................................................................................................