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Investigating the Role of DDX28 As a Novel Regulator of the Hypoxic Cap- Dependent Translation Initiation Complex

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Investigating the Role of DDX28 As a Novel Regulator of the Hypoxic Cap- Dependent Translation Initiation Complex Investigating the role of DDX28 as a novel regulator of the hypoxic cap- dependent translation initiation complex by Sonia Evagelou A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Masters of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © Sonia Evagelou, September, 2018 ABSTRACT INVESTIGATING THE ROLE OF DDX28 AS A NOVEL REGULATOR OF THE HYPOXIC CAP-DEPENDENT TRANSLATION INITIATION COMPLEX Sonia Evagelou Advisor: University of Guelph, 2018 Dr. James Uniacke The most common form of translation initiation in eukaryotes requires recruitment of the eIF4F complex to the m7GTP cap located at the 5’ end of all cellular mRNAs. eIF4F is composed of the cap-binding factor eIF4E, the RNA helicase eIF4A, and the scaffolding protein eIF4G, which together function to initiate translation. However, in response to hypoxia, a common feature of several physiological and pathological processes, eIF4E is inhibited. Hypoxic cells maintain translation of select transcripts by utilizing the eIF4E homologue, eIF4E2, as an alternative cap- binding protein as part of the eIF4FH complex. We have identified DDX28 as a novel regulator of eIF4FH by demonstrating that knocking-down DDX28 results in an overall increase in cap- bound eIF4E2 and eIF4E2-mediated translation under hypoxia. Additionally, depletion of DDX28 confers a proliferative advantage to cells grown in hypoxic conditions, which we suspect is a consequence of the translational upregulation of a subset of hypoxic mRNAs. Dedication In loving memory of my late grandmother. Brevis a naturâ nobis vita data est; at memoria bene redditæ vitæ est sempiterna. iii Acknowledgements First and foremost I would like to thank my advisor Dr. James Uniacke for his excellent mentorship over the past several years, in addition to providing me with the liberty to explore this project as an independent researcher. My development as a scientist in his lab has far exceeded what I imagined was possible in such a short period of time, and the knowledge I have gained will not only benefit me in the field of research, but in everyday life as well. I would also like to thank Erin Specker whose extensive help and generosity has contributed significantly to my positive experience in the lab. My gratitude also extends to past and present members of the lab: Sara Timpano, Dr. Phil Medeiros, Gaelan Melanson, Andrea Brumwell, Sydney Pascetta, Nicole Kelly, Joszef Varga, Brianna Guild and Olivia Bebenek for their continued assistance and friendship over the years. To my advisory committee, Dr. Ray Lu and Dr. Jnanankur Bag, thank you for your insight and advice throughout the course of my project. I would like to give a special thanks to my family for their incredible support throughout this journey, none of this would have been possible without you. Additionally, to my wonderful boyfriend Daniel Prevedel, thanks for always being a shoulder to cry on and letting me talk about my project for hours when no one else wanted to listen! iv Declaration of Work Performed Olivia Bebenek performed the hypoxic polysome profiling for RNA analysis and the normoxic and hypoxic polysome RT-PCR, and qPCR. Bromodeoxyuridine labeling experiments were performed by Erin Specker. All other experiments were performed by the author. v Table of Contents ABSTRACT .................................................................................................................................. II DEDICATION ............................................................................................................................ III ACKNOWLEDGEMENTS ....................................................................................................... IV DECLARATION OF WORK PERFORMED .......................................................................... V LIST OF FIGURES ................................................................................................................. VIII LIST OF ABBREVIATIONS .................................................................................................... IX Chapter 1. Introduction ............................................................................................................... 1 1.1 Hypoxia in Development and Disease ................................................................................. 1 1.2 The HIF Program ................................................................................................................. 2 1.3 Cap-Dependent Translation Initiation and eIF4F ................................................................ 8 1.4 mTOR and Hypoxic Regulation of eIF4F .......................................................................... 11 1.5 IRES-Mediated Translation ............................................................................................... 14 1.6 Hypoxic Cap-Dependent Translation and eIF4FH ............................................................. 15 1.7 Regulation of eIF4FH by DDX28 ....................................................................................... 17 1.8 Hypothesis and Research Objectives ................................................................................. 22 Chapter 2. Materials and Methods ............................................................................................ 24 2.1 Cell Culture and Reagents .................................................................................................. 24 2.2 Generation of Stable Cell Lines ......................................................................................... 24 2.2.1 DDX28 Knockdown Cells ....................................................................................................... 24 2.2.2 DDX28-FLAG Cells ................................................................................................................ 25 2.3 Western Blot Analysis and Antibodies .............................................................................. 25 2.4 Polysome Profiling ............................................................................................................. 26 2.4.1 Polysome Protein Analysis ...................................................................................................... 27 2.4.2 Polysome RNA Isolation, RT-PCR, and quantitative PCR ..................................................... 27 2.5 GFP-Trap Immunoprecipitation ......................................................................................... 28 2.6 FLAG Immunoprecipitation ............................................................................................... 29 2.7 Analysis of Cap-Binding Proteins ...................................................................................... 30 2.8 Crystal Violet Growth Curves ............................................................................................ 31 vi 2.9 Bromodeoxyuridine Labeling ............................................................................................ 32 Chapter 3. Results ....................................................................................................................... 33 3.1 HIF-2α mediates the interaction between DDX28 and eIF4E2 ......................................... 33 3.2 Knockdown of DDX28 enhances the cap-binding affinity of eIF4E2 ............................... 35 3.3 Knockdown of DDX28 increases eIF4E2-mediated translation ........................................ 38 3.3 Knockdown of DDX28 increases translation of EGFR mRNA ......................................... 42 3.4 Knockdown of DDX28 Increases Cell Viability in Hypoxia ............................................. 44 3.5 Knockdown of DDX28 Increases Cell Proliferation Rates in Hypoxia ............................. 46 Chapter 4. Discussion ................................................................................................................. 49 Chapter 5. Conclusions and Future Directions ........................................................................ 61 References .................................................................................................................................... 63 vii List of Figures Figure 1. Alignment of HIF-1α and HIF-2α structural domains...................................................5 Figure 2. Overview of the regulation of HIF-α in response to changing oxygen concentrations..6 Figure 3. Proposed model of the eukaryotic translation initiation complex mediating cap- dependent translation during normoxic and hypoxic conditions...................................................12 Figure 4. Immunoprecipitation of FLAG-eIF4E2 from normoxic and hypoxic U-87 MG cell lysates............................................................................................................................................18 Figure 5. Schematic diagram of the conserved motifs of the DEAD-box protein superfamily...19 Figure 6. Standard workflow of a cap-affinity assay ...................................................................22 Figure 7. Isolating polyribosomes using polysome profiling ......................................................23 Figure 8. DDX28 interacts with HIF-2α in U-87 MG .................................................................34 Figure 9. Knockdown of DDX28 increases the cap-binding affinity of eIF4E2 in U-87 MG.....36 Figure 10. Knockdown of DDX28 in U-87 MG significantly increases polysome-associated eIF4E2 in hypoxia.........................................................................................................................40
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