Thesis Master Doc Revamp

The impact of Alu elements on the human proteome Sarah Elizabeth Atkinson Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Chemistry May 2019 Acknowledgements Acknowledgements Firstly, I would like to thank Prof. Paul Taylor, Dr Michael Webb and Dr Suzanne Dilly for their guidance and support throughout my PhD, as well as the University of Leeds and Valirx Plc for providing me with this wonderful opportunity. Secondly, I would like to extend my deepest thanks to the members of Lab 1.49, past and present, for all the support they have provided throughout my PhD. In particular, I would like to thank Jack Caudwell for providing me with constant moral support and always keeping a smile on my face, as well as helping to provide the warm and social atmosphere that makes working in 1.49 so special. You made my entire PhD experience infinitely more enjoyable! To Ryan, I hope your next desk buddy is more of a pushover and your future desk take overs are more successful than they were with me. Additionally, I would like to thank Pablo, Devón and JoDo for providing me with friendship and support both inside and outside of the university. It is, of course, customary to thank my parents, who have allowed me to remain in debt with the ‘Bank of Mum and Dad’ whilst I have postponed getting a ‘real job’ for as long as physically possible. On a more serious note, they have always provided me with the upmost support and it has never gone unappreciated. Importantly, I would like to thank Dr Jennifer Miles who helped me leave my chemical roots and learn the ‘nitty gritty’ of biology. I certainly would have struggled to get my head round some of the trickier concepts without your help. I would also like to extend my gratitude to all the members of the Aspden group; in particular, Katerina who provided me with constant guidance during tissue culture and ‘polysoming’ and dedicated a large amount of time (often extending into the evening) to helping me and making me feel welcome in FBS. i Abstract Abstract Approximately 45% of the human genome is comprised of mobile, or transposable, DNA elements (TEs). Of this, 11% is attributed to Alu elements. Alu elements are approximately 300 base pairs in length and are primarily located in the introns of non-coding DNA. However, in some cases, the introduction of an alternative splice site, as a result of an Alu insertion in a protein-coding region, leads to the exonisation of a partial Alu sequence. This exonisation can lead to the expression of an alternative protein isoform which may have disrupted or altered function and therefore, could have the potential to be cause disease. Through the use of bioinformatics, this project firstly aimed to predict the extent of Alu exonisation and subsequent translation in the human proteome. Additionally, through the use of local sequence alignments, the nature of observed insertions could also be studied. Once prior aims were established, a series of techniques were used to study the possible effects of translated Alu insertions on the structure and function of proteins. A number of protein variants were expressed and purified from E. coli. Using biophysical techniques, such as ITC and CD, Alu structure and any effects of Alu insertions on the ligand binding and stability of MBP were studied. Additional binding experiments were performed as a means to explore a potential binding interaction between an Alu-like sequence with geldanamycin, an interaction which was initially observed using phage display. A secondary avenue of research was performed in collaboration with the Aspden and Wurdak groups at the University of Leeds to investigate the difference in translation levels of ‘Alu’ and ‘non-Alu’ mRNAs in human cells. Analysis was performed using a combination of polysome profiling, reverse transcription and quantitative PCR. ii Abbreviations Abbreviations AC Alu-containing AI Auto-induction AIP Acute intermittent porphyria ARMD Alu recombination-mediated deletion ASC-1 Activating signal cointegrator 1 ASCC1 Activating signal cointegrator 1 complex subunit 1 ATP Adenosine triphosphate BCAS3 Breast cancer amplified sequence 3 BCAS4 Breast cancer amplified sequence 4 BE Barrett’s esophagus BLAST Basic local alignment search tool Bp Base pair BSA Bovine serum albumin CASC5 Kinetochore scaffold 1 CBPC2 Cytosolic carboxypeptidase 2 CBPC3 Cytosolic carboxypeptidase 3 CCNJL Cyclin-J-like protein CD Circular dichroism CDKL5 Cyclin-dependent kinase like 5 cDNA Complementary DNA CDNN Circular dichroism analysis using Neural Networks CK5P1 CDK5 regulatory subunit-associated protein 1 iii Abbreviations CNTLN Centlein Cp Heat capacity CP089 UBF0764 protein C16orf89 Da Daltons DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DSB Double strand break DSC Differential scanning calorimetry dsDNA Double-stranded DNA DTT Dithiothreitol E. coli Escherichia coli EAC Esophageal adenocarcinoma EN Endonuclease ER Endoplasmic reticulum F193A Protein FAM193A FA Fluorescence anisotropy FAM Fossil Alu monomer FITC Fluoroscein isothiocyanate FLAM Free left Alu monomer FP Fluorescence polarisation FPLC Fast protein liquid chromatography FRAM Free right Alu monomer FT Flow-through FXL18 F-box/LRR-repeat protein 18 iv Abbreviations GBM1 Glioblastoma multiforme GLYG2 Glycogenin-2 GM Geldanamycin GST Glutathione-S-transferase HSP90 Heat-shock protein 90 HTP High-throughput IPTG Isopropyl-β-D-1-thiogalactopyranoside ITC Isothermal titration calorimetry ITCH E3 ubiquitin-protein ligase Itchy homolog kDa Kilodaltons LB Lysogeny broth LINE Long interspersed nuclear element LTR Long-terminal repeat M4K1 Mitogen-activated protein kinase kinase kinase kinase 1 MAGI3 Membrane-associated guanylate kinase, WW and PDZ domain- containing protein 3 MBP Maltose binding protein MKNK1 MAP kinase-interacting serine/threonine-protein kinase 1 mRNA Messenger RNA MS Mass spectrometry MTO1 Mitochondrial protein homolog MTO1 MY15B Unconventional myosin-15B MYL10 Myosin regulatory light chain 10 nAC Non-Alu-containing NADPH Nicotinamide adenine dinucleotide phosphate hydrogen v Abbreviations NCBI National Centre for Biotechnology Information NEK4 Never in mitosis gene A (NIMA)-related kinase 4 NHEJ Non-homologous end-joining Ni-NTA Nickel-nitrilotriacetic acid NPCL1 NPC1-like intracellular cholesterol transporter 1 Nrk NIMA-related kinase ORF Open reading frame PAGE Polyacrylamide gel electrophoresis PBGD Porphobilinogen deaminase PBS Phosphate buffer saline PCR Polymerase chain reaction PNK Polynucleotide kinase PPP5D1 PPP5 TPR repeat domain-containing protein 1 PRR34 Proline-rich protein 34 PTM Post-translational modification qPCR Quantitative PCR RABX5 Rab5 GDP/GTP exchange factor REL Proto-oncogene c-Rel RF Restriction-free RI Refractive index RNA Ribonucleic acid RNAPIII RNA polymerase III rRNA Ribosomal RNA RT Reverse transcriptase vi Abbreviations RU Resonance unit SDM Site-directed mutagenesis SDS Sodium dodecyl sulfate SEC Size exclusion chromatography SINE Short interspersed nuclear element SPR Surface plasmon resonance SRP Signal recognition particle ssDNA Single stranded DNA SUMO Small ubiquitin-like modifier TB Terrific broth TE Transposable element TEV Tobacco etch virus TEX11 Testis-expressed protein 11 Tm Melting temperature TMM78 Transmembrane protein 78 TPRT Target-primed reverse transcription TRIP4 Thyroid hormone receptor interactor 4 TTF1 Transcription termination factor 1 TV Transcript variant TV23C Golgi apparatus membrane protein TVP23 homolog C Ub Ubiquitin UBP19 Ubiquitin carboxyl-terminal hydrolase 19 Ulp1 Ubiquitin-like-specific protease 1 UPP Ubiquitin proteasome pathway vii Abbreviations UV Ultraviolet WT Wild-type YS049 Zinc finger protein ENSP00000375192 ZMAT1 Zinc finger matrin-type protein 1 ZN195 Zinc finger protein 195 ZN415 Zinc finger protein 415 ZNF91 Zinc finger protein 91 viii Contents Contents Acknowledgements ................................................................................................ i Abstract ................................................................................................................ ii Abbreviations ...................................................................................................... iii Contents .............................................................................................................. ix Table of Figures ................................................................................................. xiii List of Tables .................................................................................................... xvii Chapter 1 An introduction to Alu elements .................................................. 1 Overview .......................................................................................................... 1 1.1 Transposable elements ................................................................ 1 1.2 The origin of Alu elements .......................................................... 4 1.3 Alu structure ............................................................................... 7 1.4 Alu movement and replication in the genome .............................. 8 1.5 Transcription and translation of Alu Elements ........................... 12 1.6 Alu elements in humans and their relation to disease ................. 16 1.7 Project aims .............................................................................. 18 Chapter 2 Bioinformatic analysis of Alu elements ....................................

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