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UNDERSTANDING the ROLE of SPLICING FACTORS in CENTRIOLE DUPLICATION by Elizabeth Michelle Park a Dissertation Submitted to John

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UNDERSTANDING THE ROLE OF SPLICING FACTORS IN CENTRIOLE DUPLICATION by Elizabeth Michelle Park A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland January 2020 © 2020 Elizabeth Park All rights reserved Abstract The centriole is a microtubule-based structure that forms the core of the centrosome, the major microtubule organizing center of the cell. Each centrosome contains precisely two centrioles that are surrounded by pericentriolar material that nucleates microtubules and plays crucial roles in the cell. Cycling cells undergo exactly one round of centriole duplication that is regulated numerically, spatially, and temporally alongside the duplication of the cell’s DNA. This regulation is important for cell health and viability, as aberrations in centriole number can lead to diseases such as cancer. The complete molecular mechanisms regulating centriole duplication have not yet been discovered. Recent work identified that a subset of splicing factors is required for centriole biogenesis. This revealed a previously unstudied means of post-transcriptional regulation that centriole proteins could undergo to ensure that the centriolar building blocks are translated in stoichiometrically appropriate amounts. While this subset of splicing factors was identified as playing a role in centriole duplication, the precise mechanism by which they regulate centriole biogenesis at the post transcriptional level was not identified. I sought to identify novel means of regulating centriole duplication. I uncovered an additional splicing factor, WBP11, that upon depletion, yields similar phenotypes to the depletion of the fourteen other splicing factors implicated in centriole biogenesis. I found that WBP11, SNW1, and likely other centriole-related splicing factors are required to splice out short, weak introns such as those found at the 3’ terminus of the TUBGCP6 pre-mRNA. Improper splicing of this ii transcript leads to reduced protein levels, failed centriole biogenesis, and mitotic defects that lead to cell death. This is the first mechanistic link between a subset of splicing factors and centriole biogenesis. This finding could have clinical implications beyond my work, as splicing factors have been shown to play direct roles in cancer progression and tumorigenesis. Some splicing factors have also been implicated in microcephaly, a disease hallmarked primarily by centriole defects and mutations in the structural components of the centriole. Understanding how splicing factors regulate protein levels and the role that intron retention could play in post-transcriptional control could provide valuable insights to both cancer and microcephaly. iii Thesis advisor and primary reader: Dr. Andrew J. Holland Thesis committee member and secondary reader: Dr. Carolyn Machamer iv Acknowledgements First and foremost, I would like to thank my mentor, Dr. Andrew Holland. Andrew is an admirably passionate, world-class scientist whose love for molecular biology and excitement for new data is unparalleled. He radiates an infectious enthusiasm for repeatable and sound data that spreads through the lab to all of us. He gives everything to his students and is always around to provide mentorship and guidance. Andrew has an incredible ability to see the big picture while simultaneously knowing the little details that need to make up that big picture, and that translates into his mentoring to allow us to learn what the next steps should be and how to synthesize a project. Although a younger PI, there was never any question as to whether or not Andrew would be successful as a mentor, and I am very thankful to have had his guidance and motivation over the past few years. I hope that as I continue in science, I can take away even half of the enthusiasm and excitement that Andrew brings to lab every day. I am incredibly grateful to have been taught rigorous scientific methods, clear and concise methods of conveying my findings to others, and perseverance even when experiments are failing more often than succeeding. Those lessons as a scientist are invaluable, and I owe them to Andrew. I finally have to thank Andrew for his unwavering support, not only during my project but also during my career search and acceptance of my dream job. He is an incredible mentor to his students, and I feel very fortunate to have spent the past few years learning from him. In addition to rigorous science, Andrew has cultivated a lab environment that is incredibly supportive and a true joy to be a part of. I owe a special thanks to my labmates, both past and present, for being incredible friends, support systems, and coffee buddies. They always say you v should surround yourself with people who are smarter than you, and that is precisely why I joined Andrew’s lab. Michelle, Bram, and Tyler, Andrew’s first three graduate students, were an incredible fountain of information to learn from and, importantly, were a great trio of friends to spend hours in lab alongside every day. Lauren and Thao brought even more intelligence and camaraderie into the lab, and I am so thankful to have their friendships. They have been amazing scientists to bounce ideas off of and great friends to relax with after lab. Finally, Phillip, Colin, and Gina are a wonderful new set of students who I know will contribute amazing things to the Holland lab and centriole field. Phillip, in addition to teaching me the ropes as the technician of the lab at the time of my joining, is also owed a thanks for educating me about movies and allowing me to make successful pop culture references. Training at Johns Hopkins has been an incredible experience – I can see why it is the “dream school” for so many people, and I am so fortunate to have been able to live out the dream. Being in such a collaborative environment fostered a wonderful place to develop as a scientist. I owe a debt of thanks to Drs. Sarah Wheelan, Liliana Florea, Anuj Gupta, Amir Rattner, Jacob Heng, and Boris Zinshteyn for their assistance with my RNA-Seq experiments and learning how to code. I could not have completed this work without them. They spent countless hours with me helping me understand the coding component of my work, and without their assistance my project would have proceeded much more slowly. I appreciate all of the time spent correcting my vast number of code errors, their sharing of programs, and their willingness to make themselves available for me. I would also like to thank Drs. Carol Greider and Mary Armanios, who at the beginning of my time in the Holland lab, provided useful insights at the start of my project during our group lab meetings as well as sharing lab equipment. During the final couple years of vi my PhD, Drs. Rachel Green and Geraldine Seydoux provided access to shared lab equipment without which I could not have done my project. My thesis committee members, Drs. Carolyn Machamer, Brendan Cormack, and Debbie Andrew, have been instrumental in my success as a PhD student. My meetings were always constructive both scientifically and professionally. I especially appreciate Brendan’s willingness to provide career advice as I finished up my graduate studies. They say it takes a village to get a PhD, and I am very fortunate to have an amazing village helping me along the way. My friends in graduate school have been sources of unwavering support and kindness throughout the years. It has been such a pleasure bouncing scientific ideas off of them as well as blowing off steam outside of lab and enjoying Baltimore with them. I also have to thank my non-graduate friends, whether from college, grade school, or travel friends I’ve picked up along the way, I have had such great fortune in building my village and could not be luckier to have had so many supportive people backing me along the way. Finally, this section would not be complete without thanking my most avid supporters, my family. During graduate school, my family grew by one four-legged member, and Juneau has been the best thing that ever happened to me. I am so thankful to have rescued her, and coming home to her every day after lab was always the highlight of my day. Thank you to Juneau for providing me with plenty of tail wags and puppy kisses. As for my parents, they have always stood beside me no matter what, and I know they couldn’t be prouder. Thank you for taking all of my stressed phone calls, allowing me to practice the occasional presentation with you, providing me with chocolate from time to time, and supporting me through every single dream vii that I dare to take. There are not enough words to express my gratitude. I could not have done this without you. viii Table of Contents Abstract................................................................................................................................. ii Acknowledgements ................................................................................................................ v Table of Contents ................................................................................................................. ix List of Figures ...................................................................................................................... xi Chapter 1: Introduction ......................................................................................................... 1 1.1 Overview of the centrosome ....................................................................................................1
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