NOVEL PROPERTIES OF HNRNP-UL1: ITS POSSIBLE ROLE IN THE PATHOGENESIS OF ALS. By KENNY MATTHEW PRATT A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Cancer and Genomic Sciences The Medical School University of Birmingham October 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Heterogeneous nuclear ribonucleoprotein-U like 1 (hnRNP-UL1) is a protein with numerous roles within the cell, including RNA processing and responses to DNA damage. Within this study two novel aspects of the protein are explored: the role of a putative nucleotide-binding domain and the protein’s possible involvement in amyotrophic lateral sclerosis (ALS). hnRNP-UL1 is known to have a putative nucleotide-binding domain within its central region containing both a Walker A and Walker B motif. This region had not been investigated previously and was therefore of great interest in this study. The Walker A motif was shown to bind adenosine triphosphate (ATP) and the region appears to possess protein kinase activity. A biological substrate and function for these activities were not established, but these observations suggest that there are still layers of complexity to hnRNP-UL1’s cellular roles to be elucidated. ALS is a late-onset neurodegenerative disease with limited treatment strategies and poor patient outcomes. Many of the proteins involved in its pathogenesis have two properties in common: they have roles in RNA-processing and possess prion-like domains (PrLDs). The properties of hnRNP-UL1 appertain to both of these and therefore it was of great interest when ALS patients were discovered with heterozygous hnRNP-UL1 mutations. Results showed that cells possessing the ALS patient mutations (R639C and R468C) had no DNA damage response (DDR) defects or mislocalisation of the protein, but their ssDNA/RNA- binding capability was markedly reduced. Whilst no direct causative links to ALS pathogenesis were shown with the hnRNP-UL1 patient mutations in this study, growing evidence implies good reason for the protein to have involvement in the disease. ACKNOWLEDGEMENTS I am extremely grateful to my supervisor, Roger, for his continued help and advice in all aspects of the work I have undertaken as part of my PhD. He is responsible for me coming to Birmingham to pursue working in science and I am very glad to have completed my research under his experienced and nurturing tutorage. There are also many other members of staff that I would like to thank for their help and friendship. Andy provided good advice and guidance on the project, as well as daily sport-related chats. I am also thankful to Grant for his advice and knowledge, plus reagents and equipment. I must thank Elizabeth, Sally, Ruth, Helen, Kirsty, Alex, Tom, John, Martin, Phil and Rob for help along the way, and particularly Ruth and Elizabeth for technical help with certain procedures. I also thank the whole IBR 1st floor who are a great bunch of people and it is a welcoming and friendly place to work. I am also thankful for the financial support given to me by the University of Birmingham Medical School and the Medical Research Council. Finally, I would like to thank every single member of my family for supporting me from when I was young right up until this point, especially my Ma and Pa. I started my own family whilst doing this PhD and nothing has kept me happier during this time in my life than my wife Hannah and son Henry. Coming home to play with Henry helped to relieve stress and put in to perspective a failed experiment. Finally, thank you Hannah for all the love, commitment and sacrifices made in the past four years. TABLE OF CONTENTS I INTRODUCTION ........................................................................................................... 1 1.1 The DNA molecule ...................................................................................................... 2 1.2 The DNA damage response (DDR).............................................................................. 4 1.2.1 Single strand break (SSB) repair ......................................................................... 8 1.2.2 Base excision repair (BER) ............................................................................... 10 1.2.3 Nucleotide excision repair (NER) ..................................................................... 11 1.2.4 Non-homologous end joining (NHEJ) .............................................................. 11 1.2.5 Homologous recombination (HR)..................................................................... 13 1.2.6 Diseases associated with malfunction of the DDR ........................................... 16 1.3 Heteronuclear ribonucleoproteins (hnRNPs) ............................................................. 24 1.3.1 hnRNPs ............................................................................................................. 24 1.3.2 hnRNP-UL1 ...................................................................................................... 26 1.3.3 hnRNPs in the DDR .......................................................................................... 30 1.3.4 hnRNP-UL1 in the DDR ................................................................................... 33 1.3.5 hnRNP-UL1’s Walker A motif and activity of PNKP...................................... 36 1.4 Amyotrophic lateral sclerosis (ALS) .......................................................................... 42 1.4.1 ALS aetiology and pathogenesis ....................................................................... 43 1.4.2 RNA processing and ALS ................................................................................. 49 1.4.3 Prion-like domain (PrLD) proteins and ALS .................................................... 53 1.4.4 ALS and the DDR ............................................................................................. 57 1.5 Aims ........................................................................................................................... 62 II MATERIALS AND METHODS ................................................................................. 63 2.1 Cell biology techniques .............................................................................................. 64 2.1.1 Cell lines ........................................................................................................... 64 2.1.2 Cell culture media ............................................................................................. 64 2.1.3 Maintenance and passage of cell lines .............................................................. 64 2.1.4 Cryopreservation of cell lines ........................................................................... 65 2.1.5 IR and UV irradiation ....................................................................................... 65 2.1.6 Drug treatments ................................................................................................. 66 2.1.6.1 Camptothecin ............................................................................................... 66 2.1.6.2 Kinase inhibitors .......................................................................................... 66 2.1.6.3 Tert-butylhydroquinone ............................................................................... 67 2.1.7 RNA interference (RNAi) ................................................................................. 67 2.1.8 Transfection of plasmid DNA ........................................................................... 68 2.2 Molecular biology techniques .................................................................................... 69 2.2.1 Preparation of media and plates ........................................................................ 69 2.2.2 Bacterial transformations .................................................................................. 69 2.2.3 Large scale preparation of DNA plasmids ........................................................ 70 2.2.4 Agarose gel electrophoresis .............................................................................. 71 2.2.5 Site-directed mutagenesis PCR ......................................................................... 72 2.2.6 DNA sequencing ............................................................................................... 73 2.2.7 Cloning .............................................................................................................. 74 2.3 Protein and DNA biochemistry techniques ................................................................ 75 2.3.1 Preparation of total cell lysates ......................................................................... 75 2.3.2 Preparation of lysates from cells for co-immunoprecipitation ......................... 76 2.3.3 Determination of protein concentration ............................................................ 76 2.3.4 SDS-polyacrylamide gel