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An Interplay Between Nonsense-Mediated Decay and DNA Damage Response Pathways

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An Interplay Between Nonsense-Mediated Decay and DNA Damage Response Pathways An interplay between nonsense-mediated decay and DNA damage response pathways Fatemeh Ghasemi Master Thesis Department of Biosciences Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO June 2020 © Fatemeh Ghasemi June 2020 An interplay between nonsense-mediated decay and DNA damage response pathways Supervisor: Rafal Ciosk Co-supervisors: Pooja Kumari, Yanwu Guo http://www.duo.uio.no/ Trykk: Reprosentralen, Universitetet i Oslo II Acknowledgement The work presented in this master thesis was carried out at the Department of Biosciences, University of Oslo in the period between April 2019 to June 2020. First and foremost, I’d like to thank my supervisor Rafal Ciosk for giving me the opportunity to work in his group. Thank you for all your help and positivity. I greatly appreciate everything I learned in my time here. Second, I’d like to express my deep gratitude to my co-supervisor Pooja Kumari, without whom I couldn’t have done this. Thank you for your daily guidance and support in the lab, I truly appreciate all the advice you’ve given me. Further, I’d like to thank everyone else in the Ciosk group, especially Yanwu Guo, for all their practical help in the lab, and for writing this thesis. Your input and advice have been greatly appreciated. Divya and Melanie, thank you for cheering me up every single day. Thank you also to all my friends in the Falnes group who helped me out when I was wandering the hallway looking lost. Lastly, I would like to thank my parents for their unending love and support, and for believing in me. I also want to express my gratitude to all my amazing friends who made my years in Oslo so wonderful and unforgettable. III Table of contents Acknowledgement .................................................................................................................... III Table of contents ...................................................................................................................... IV 1 Introduction ........................................................................................................................ 5 1.1 Nonsense-mediated mRNA decay (NMD) .................................................................. 5 1.1.1 Function of the NMD pathway............................................................................. 5 1.1.2 Mechanism ........................................................................................................... 5 1.1.3 Project background ............................................................................................... 8 1.2 DNA damage ............................................................................................................... 9 1.2.1 Genetic integrity ................................................................................................... 9 1.2.2 Sources of DNA damage ...................................................................................... 9 1.2.3 Types of DNA damage ....................................................................................... 10 1.2.3.1 Double-strand breaks (DSB) .......................................................................... 11 1.2.4 DNA damage response (DDR) ........................................................................... 12 1.3 DNA repair ................................................................................................................ 14 1.3.1 Direct reversal .................................................................................................... 14 1.3.2 Repair of single strand damage .......................................................................... 15 1.3.3 Repair of double-strand breaks .......................................................................... 15 1.4 Caenorhabditis elegans ............................................................................................. 21 1.4.1 C. elegans as a model organism ......................................................................... 21 1.4.2 C. elegans as a DNA repair model ..................................................................... 23 1.4.3 DNA damage repair in C. elegans ..................................................................... 24 1.5 Project objectives ....................................................................................................... 25 2 Materials and Methods ..................................................................................................... 26 2.1 Maintenance of C. elegans ........................................................................................ 26 2.1.1 Strains and constructs ......................................................................................... 26 2.1.2 Synchronization .................................................................................................. 27 2.2 C. elegans genetic methods ....................................................................................... 27 2.2.1 Worm lysis ......................................................................................................... 27 2.2.2 LacZ staining ...................................................................................................... 28 IV 2.2.3 Generating males ................................................................................................ 29 2.2.4 Genetic crossover to create double mutants ....................................................... 29 2.3 Characterization and phenotyping ............................................................................. 29 2.3.1 Developmental assay .......................................................................................... 29 2.4 Stress induction.......................................................................................................... 29 2.4.1 IR assay .............................................................................................................. 29 2.4.2 Reporter construct heat shock ............................................................................ 30 2.5 RNA interference (RNAi) ......................................................................................... 32 2.6 Molecular experiments .............................................................................................. 33 2.6.1 Polymerase chain reaction (PCR) ...................................................................... 33 2.6.2 Electrophoresis ................................................................................................... 34 2.6.3 Cloning ............................................................................................................... 35 2.7 RT-qPCR ................................................................................................................... 36 2.7.1 Isolation of total RNA using TRIzol reagent ..................................................... 36 2.7.2 cDNA synthesis .................................................................................................. 37 2.7.3 Reverse transcription quantitative PCR (RT-qPCR) .......................................... 39 2.8 Microscopy ................................................................................................................ 41 3 Results .............................................................................................................................. 42 3.1 smg-1(tm849) and smg-2(tm6028) are sensitive to ionizing radiation (IR) ............... 42 3.2 Characterization of smg-1(tm849) and smg-2(tm6028) knockout mutants ............... 43 3.3 Knockdown of DDR pathway genes in smg-1(tm849) and smg-2(tm6028) ............. 46 3.4 Homologous recombination repair increases when smg-1 and smg-2 are knocked down 48 3.5 smg-1 and smg-2 are upregulated after irradiation .................................................... 50 4 Discussion ........................................................................................................................ 51 4.1 Localization of NMD components ............................................................................ 51 4.2 Hypersensitivity of NMD mutants to IR ................................................................... 51 4.3 Change in expression levels of smg genes upon irradiation ...................................... 52 4.4 Connection to DNA damage response network ........................................................ 53 5 Conclusion ........................................................................................................................ 55 6 Future Prospects ............................................................................................................... 56 7 References ........................................................................................................................ 57 8 Appendix .......................................................................................................................... 62 V 8.1 Buffer and media ....................................................................................................... 62 8.2 Standard cloning protocol .......................................................................................... 67 8.3 Raw data .................................................................................................................... 76 8.3.1 Developmental assay .......................................................................................... 76 8.3.2 Survival assay ....................................................................................................
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