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Characterizing G-Quadruplex Mediated Regulation of the Amyloid Precursor Protein Expression by Ezekiel Matthew David Crenshaw Oc

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I Characterizing G-Quadruplex Mediated Regulation of the Amyloid Precursor Protein Expression By Ezekiel Matthew David Crenshaw October, 2016 A Dissertation Presented to the Faculty of Drexel University Department of Biology In partial fulfillment of the Requirements for the Degree of Ph.D. Daniel Marenda, Ph.D. (Chair) Elias Spiliotis, Ph.D. Associate Professor, Associate Professor, Director, Graduate Student Program Director, Cell Imaging Center Co-Director, Cell Imaging Center Dept. Biology Dept. Biology Drexel University Drexel University Tali Gidalevitz, Ph.D. Aleister Saunders, Ph.D. (Advisor) Assistant Professor, Dept. Biology Associate Professor, Drexel University Director of the RNAi Resource Center, Dept. Biology Jeff Twiss, Ph.D. Senior Vice Provost for Research, Drexel University Professor, SmartState Chair, Childhood Neurotherapeutics Michael Akins, Ph.D.(Co-Advisor) Dept. Biology University of South Carolina Assistant Professor, Dept. Biology Drexel University II Acknowledgements Proverbs 3:5-6, “Trust in the Lord with all thine heart and lean not unto thine own understanding. In all thine ways acknowledge Him and He shall direct thy paths” I want to start off my acknowledging my LORD, who is known by many names. He is El Elyon, The Most High God; He is YAHWEH Shammah, for He has been there for me; He is YAHWEH Yireh, for He has provided for all of my needs; He is YAHWEH Shalom, for He is my peace. He is Yeshua Ha Mashiach, for He is my Savior. Coming from humble beginnings, my faith in the LORD has been the source of strength and encouragement to overcome the many obstacles I faced growing up, as well as in my pursuit for my Ph.D. I’d like to thank my advisor, Dr. Aleister Saunders, for bringing me into his lab and for his help and support during the past 5 years. I know I had struggles in the lab as far as research, but I am thankful to him for giving me an opportunity to grow and learn as a graduate student and as young scientist. I am especially thankful that Aleister allowed me to explore other opportunities outside of the lab such as teaching both locally and internationally, research conferences, additional courses, and professional development workshops. I want to thank my co-advisor, Dr. Michael Akins who has been very instrumental in moving my thesis work forward. Michael brought me into his lab as a joint student and I was able to learn various techniques from members in his lab that really help me in my own research. I appreciate the time he took to mentor me and just for his enthusiasm to advance scientific research. I’d like to thank my thesis committee Drs. Daniel Marenda, Elias Spiliotis, Tali Gidalevitz, and Jeff Twiss for their guidance and helpful suggestions related to my research, as well as in courses and seminars. III Next, I want to thank Drs. Haizhi Wang and Michal Sharoni for their help and support. I really felt that we were a small lab family. We struggled and celebrated together and created an atmosphere that was still fun and exciting to conduct research. It has meant so much to me for them to take the time out of their busy schedules to teach me a technique or listen to me give a search talk and provide me with feedback that didn’t make me feel like I was not qualified to be a graduate student. I also want to thank the previous lab members Drs. Suruchi Utreja and Anna Vorobyeva for helping me when I initially joined the lab, as well as Brendan Mullen, Angela Tuckowski and Gavi Horwitz for their help. Finally, I’d like to thank my wife, Elisha Crenshaw and our children, Israel, Zachariah, and Elaya for their continued love and support. It meant so much to me to come home to smiles and encouragement from them. When I felt like giving up or that I wasn’t qualified to pursue a doctoral degree, they believed in me, prayed for me, as well as laughed and cried with me. I am grateful because now our children can grow up and realize that they too can follow their dreams. IV Table of Contents List of Tables……………………………………………………………………………VII List of Illustrations ……………………………………………………………….…….VIII Abstract ........................................................................................................................................... IX Chapter 1: Introduction ................................................................................................................... 1 BACKGROUND AND SIGNIFICANCE ................................................................................... 1 The Biology of APP ..................................................................................................................... 4 Regulation of APP Gene Expression ........................................................................................... 7 Summary .................................................................................................................................... 14 Figure Legends .......................................................................................................................... 16 Chapter 2........................................................................................................................................ 20 Abstract ...................................................................................................................................... 21 Introduction ................................................................................................................................ 22 Materials and Methods:................................................................................................................. 24 APP G-quadruplex identification and sequence conservation ................................................... 24 RNA preparation ........................................................................................................................ 25 Circular Dichroism (CD) Spectroscopy ..................................................................................... 25 Data fitting ................................................................................................................................. 26 Constructs and Site-Directed-Mutagenesis ................................................................................ 27 Cell Culture and Transfection .................................................................................................... 28 Metabolic Labeling using L-azidohomoalaine (AHA) .............................................................. 29 Immunoprecipitation .................................................................................................................. 29 Click Chemistry Reaction .......................................................................................................... 30 Western Blot .............................................................................................................................. 30 Luciferase Assays ...................................................................................................................... 31 RNA isolation and Real Time PCR ........................................................................................... 31 Aβ ELISA .................................................................................................................................. 32 Statistical Analysis ..................................................................................................................... 32 Results ............................................................................................................................................ 33 Bioinformatic identification and sequence conservation of an APP 3’UTR G-quadruplex ...... 33 Structural Confirmation of the APP 3’UTR Putative G-quadruplex ......................................... 34 V The APP 3’UTR G-quadruplex does not regulate expression levels of the APP RNA ............. 35 The APP 3’UTR G-quadruplex Negatively Regulates APP Protein Levels .............................. 36 Translational Control of APP by G-quadruplex ........................................................................ 38 Discussion ...................................................................................................................................... 39 Acknowledgements........................................................................................................................ 41 Figure Legends ............................................................................................................................... 42 Chapter 3........................................................................................................................................ 50 Abstract ...................................................................................................................................... 50 Introduction ................................................................................................................................ 51 Overview of Translation ............................................................................................................ 52 Importance of the 3’UTR ........................................................................................................... 54 Regulation of APP by 3’UTR RNA Binding Proteins ............................................................... 55 Regulation of APP by microRNAs ............................................................................................ 57 Regulation of APP by Guanine
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