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First Line of Title RNA-BINDING PROTEIN MEDIATED POST-TRANSCRIPTIONAL CONTROL OF GENE EXPRESSION IN EYE DEVELOPMENT AND DISEASE by Soma Dash A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Sciences Spring 2018 © 2018 Dash All Rights Reserved RNA-BINDING PROTEIN MEDIATED POST-TRANSCRIPTIONAL CONTROL OF GENE EXPRESSION IN EYE DEVELOPMENT AND DISEASE by Soma Dash Approved: __________________________________________________________ E. Fidelma Boyd, Ph.D. Chair of the Department of Biological Sciences Approved: __________________________________________________________ George H. Watson, Ph.D. Dean of the College of Arts and Sciences Approved: __________________________________________________________ Ann L. Ardis, Ph.D. Senior Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Salil A. Lachke, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Melinda K. Duncan, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Robert W. Mason, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Jia L. Song, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Donna S. Woulfe, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS First, thank you Dr. Lachke. I am most thankful for all the opportunities that you have provided me through the five years. None of this would have been possible if not for your foresight, planning, collaborations and relentless training and mentoring. Of all things, you have lead by example. And for reminding me that hard work always pays. Dr. Melinda Duncan, thank you for all the guidance you have provided me. But most of all, I am amazed by how you have always made time for all the graduate students who wanted to talk to you and how no topic is off limit with you. Dr. Jia Song, Dr. Robert Mason and Dr. Donna Woulfe, thank you for all your contributions in my committee meetings and for keeping me on my toes. I am still learning how to defend my thought process and I am getting better because of all your support. Dr. Patricia DeLeon, thank you for providing me the first opportunity to explore myself as a bench scientist when I joined UD as a Professional Science Masters Student. You took me on as a volunteer and trusted me with experiments required to finish two manuscripts, which solidified my plans for pursuing a doctoral degree. Department of Biology… In the last 5 years I have asked for help from almost everyone in the department, professors, students, staff and I have been given help generously. v My lab members Carrie and Archana have made significant contributions to both my professional and personal life. Carrie has been my sounding board and the person who reminded me not to be full of myself. Archana, my partner in crime, I will always cherish all our crimes and fun we had along the way. Sandeep, Shaili, Salma, Kimia you have been awesome labmates. From intellectual to most insipid discussions, you have made lab a fun place. I cannot imagine my life UD without my support system here in the form of my friends Dr. Aasma Khan, Dr. Sona Lakshme Balasubramaniam, Anagha Kulkarni, Dr. Devesh Radhakrishnan, Dr. Hemanth Akkiraju and Dr. Abhishek Sahasrabudhe. Aasma taught me everything I needed to know before I actually started work in my lab: cell culture, immunoprecipitation and cloning. For the first two years of my PhD, I would be seen more in Woulfe lab than in my lab. Whenever I had to bounce ideas or troubleshoot, I would go in search of her. Apart from the Science, she is a dear friend who has done her best to keep me sane through my PhD. Sona, Hemanth and Devesh have always supported me irrespective of the situation, be it random “I want to eat sushi” or “I don’t understand why my experiment didn’t work”. Anagha, thank you for all the laughs. Abhishek, all the credit for my working cloning experiment goes to you. Apart from them I want to acknowledge my friends from the Batatas and Ultimate group. I saw a little sun because of all of you. Sumedha, you have been my unofficial therapist. Apart from getting too much detail of my life in US, you also had the unique opportunity (disadvantage) of listening to all my miseries. That you respond to each of my narcissistic rant with patience and understanding, makes me feel like a petulant child and most blessed human at the same time. vi I thank the staff at Bioimaging facility, specially Deborah Powell for teaching me SEM imaging and Michael Moore for teaching me Confocal imaging. Thank you, biology administrative staff, especially Betty Cowgill for answering all my miscellaneous questions at all random times throughout the time I have spent here. I also thank all the professors for whom I have been a teaching assistant. I gained new experience every semester and I will carry this experience with me to my future career. At last, I thank the people responsible for my existence, my parents for their continuous support throughout my life. Papa keeps reminding me to make use of my wings and take flight. And Mama you keep me grounded, reminding me to do the necessary, prioritize. While Mom and Dad know roughly about my triumphs and challenges, my sister Ruma knows all the unnecessary details. You have supported me in every way possible reminding me to have fun along the way. Thank you for being you. vii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... xii LIST OF FIGURES ..................................................................................................... xiii ABSTRACT .............................................................................................................. xxiii Chapter 1 INTRODUCTION .............................................................................................. 1 1.1 Eye Development in Mammals ................................................................. 1 1.2 Transcriptional Regulation of Early Eye Development ............................ 5 1.3 Signaling Pathway in Early Eye Development ......................................... 8 1.4 Cellular Features That Suggest the Requirement of Post- Transcriptional Control in the Eye .......................................................... 10 1.4.1 Evidence for Cell-Specific Translational Control in the Lens .... 11 1.4.2 Evidence for mRNA Decay in Lens Development ..................... 13 1.4.3 miRNA Regulated Post-Transcriptional Regulation in the Lens 13 1.4.4 The function of RNA Granules and potentially RNA Regulons in Lens Fiber Differentiation ....................................................... 14 1.5 Discovery of Genes Linked to Eye Disorders ......................................... 19 1.6 Identification of New RNA-Binding Proteins Using iSyTE ................... 21 1.6.1 Rbm24 as a promising candidate in vertebrate eye development ................................................................................ 21 1.6.1.1 Function of Rbm24 in Zebrafish .................................. 21 1.6.1.2 Function of Rbm24 in Xenopus ................................... 23 1.6.1.3 Function of Rbm24 in Mouse ....................................... 23 1.6.1.4 Transcriptional regulation of Rbm24 expression ......... 23 1.6.1.5 Molecular function of Rbm24 ...................................... 25 1.6.2 Caprin2 as a promising candidate gene required for eye development ................................................................................ 25 viii 2 MATERIALS AND METHODS ..................................................................... 28 2.1 Animals .................................................................................................... 28 2.1.1 Generation of Rbm24 target deletion knockout mouse ............... 28 2.1.2 Generation of Caprin2 target deletion knockout mouse .............. 28 2.2 Genotyping Rbm24-/- and Caprin2cKO/cKO Mice....................................... 30 2.3 Zebrafish Maintenance ............................................................................ 31 2.3.1 Knockdown of rbm24a in zebrafish by morpholinos .................. 31 2.3.2 CRISPR/Cas9 Gene Targeting .................................................... 32 2.4 Morphological Analysis .........................................................................
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