THE CATARACT-ASSOCIATED RNA-BINDING PROTEIN CELF1 POST-TRANSCRIPTIONALLY REGULATES THE KEY EYE TRANSCRIPTION FACTOR PAX6 IN LENS DEVELOPMENT by Bailey A. T. Weatherbee A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Honors Bachelor of Science in Biological Science with Distinction Spring 2019 © 2019 Weatherbee All Rights Reserved THE CATARACT-ASSOCIATED RNA-BINDING PROTEIN CELF1 POST-TRANSCRIPTIONALLY REGULATES THE KEY EYE TRANSCRIPTION FACTOR PAX6 IN LENS DEVELOPMENT by Bailey A. T. Weatherbee Approved: __________________________________________________________ Salil A. Lachke, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Melinda K. Duncan, Ph.D. Committee member from the Department of Department Name Approved: __________________________________________________________ Gary Laverty, Ph.D. Committee member from the Board of Senior Thesis Readers Approved: __________________________________________________________ Earl Lee II, Ph.D. Director, University Honors Program ACKNOWLEDGMENTS First, I would like to thank Dr. Salil Lachke for his support, encouragement, and lessons throughout my three years in his laboratory. His work ethic, excitement for science, and continuous support have provided such a positive undergraduate research experience for me. He has trusted me with complex and exciting research and guided me through key decisions for my future. He has provided an incredible example of a humble, hard-working, inspiring scientist that I am so lucky to have learned from. I would like to thank the graduate students from the laboratory who have helped to train me, to teach me how to think like a scientist, and who have become my life-long friends. Dr. Archana Siddam truly shaped the way that I think about science. She molded me in the last year of her Ph.D., and I am forever grateful. Sandeep Aryal, since her graduation, has guided me. I am grateful for how our friendship has grown, his constant support, and his belief in my abilities. I would like Dr. Melinda Duncan and Dr. Gary Laverty for serving on my committee. Their commitment to undergraduate research and student success is extremely admirable. I would like to thank my parents for their unwavering support for my ambitions and interest in science since I was young. Palangga ko ikaw. Lastly, I would like to thank the faculty and students of the department. I joined this department as a junior in high school. Three labs and six years later, I truly can’t believe this part of my journey is ending. This culture and its people have been formative for me, professionally and personally. Thank you. iii TABLE OF CONTENTS LIST OF TABLES ........................................................................................................ vi LIST OF FIGURES ...................................................................................................... vii ABSTRACT ................................................................................................................ xvi 1 INTRODUCTION .............................................................................................. 1 1.1 The Mammalian Lens and its Development .............................................. 1 1.1.1 Structure and Function of the Ocular Lens .................................... 1 1.1.2 Cataract .......................................................................................... 3 1.1.3 Vertebrate Ocular Lens Development ........................................... 4 1.1.4 Lens Fiber Cell Differentiation ..................................................... 7 1.2 Pax6: A Key Regulator of Eye Development .......................................... 10 1.3 iSyTE: A Systems Tool for Predicting Genes that Function in Lens Development ........................................................................................... 16 1.4 Post-Transcriptional Control of Gene Expression in Lens Development and Disease ....................................................................... 18 1.4.1 RBP-mediated PTC in Lens Development .................................. 19 1.5 Celf1 Functions as a Post-Transcriptional Regulator .............................. 22 1.5.1 Celf1 Function in Alternative Splicing ........................................ 22 1.5.2 Celf1 Function in mRNA Localization ....................................... 23 1.5.3 Celf1 Function in mRNA decay .................................................. 23 1.5.4 Celf1 Function in Translation ...................................................... 24 1.5.5 Celf1 in Development and Disease ............................................. 25 1.6 Celf1 Functions Coordinately with other RBPs, including Elavl1, to Regulate Gene Expression ....................................................................... 30 2 MATERIALS AND METHODS ..................................................................... 32 2.1 Animal Husbandry ................................................................................... 32 2.2 DNA Isolation and Genotyping ............................................................... 33 2.3 Immunofluorescence ............................................................................... 34 2.4 Western Blot ............................................................................................ 34 2.5 RNA Isolation and Reverse Transcriptase Quantitative PCR ................. 35 2.6 RNA Immunoprecipitation (RIP) Assay ................................................. 36 2.7 Cell Culture ............................................................................................. 37 2.8 Celf1 Knock-Down and Overexpression Cell Lines ............................... 37 iv 2.9 Luciferase Reporter Assays ..................................................................... 38 3 RESULTS ......................................................................................................... 40 3.1 Pax6 Protein, but not RNA, is Mis-Expressed in Celf1 Knock-out Lenses ...................................................................................................... 40 3.2 Celf1 Protein Binds Pax6 RNA at the 3’ Untranslated Region ............... 44 3.3 Celf1 Exerts Translational Control over Pax6 by Binding to its 3’UTR 48 3.4 Co-immunostaining Reveals Potential Celf1 and Elavl1 Colocalization 54 3.5 Elavl1 Binds Pax6 RNA in the Lens ....................................................... 58 4 DISCUSSION ................................................................................................... 60 4.1 Conclusions ............................................................................................. 60 4.2 Future Directions ..................................................................................... 67 REFERENCES ............................................................................................................. 70 SUPPLEMENTARY METHODS .................................................................... 84 v LIST OF TABLES Supplementary Table 1: Celf1cKO/lacZKI Genotyping Primers ........................................ 84 Supplementary Table 2: RT-PCR Primers ................................................................... 84 Supplementary Table 3: Immunofluorescent Conditions ............................................. 84 Supplementary Table 4: Western Blot Conditions ....................................................... 85 vi LIST OF FIGURES Figure 1.1: Structure of the eye. The camera-type eye of vertebrates consists of the cornea and lens in the anterior portion, and the retina in the posterior. The lens and cornea are transparent tissues that function to refract and focus light onto the retina. Figure from Dash et al., 2016. ........................ 2 Figure 1.1.2: Schematic of lens fiber cell differentiation. Development of the mouse lens begins at E9.5 when the lens placode thickens following contact with the optic vesicle. The optic vesicle and the lens pit coordinately invaginate, leading to the detachment and formation of the lens vesicle. Anterior cells of the vesicle form the anterior epithelium while the posteriorly localized cells differentiate to lens fiber cells. Throughout life, the epithelial cells in the equatorial region exit the cell cycle and differentiate into secondary lens fiber cells. LP: Lens Placode, OV: Optic Vesicle, LPT: Lens Pit, OC: Optic Cup, LV: Lens Vesicle, C: Cornea, AE: Anterior Epithelium, RT: Retina, PF: Primary Fiber cell, SF: Secondary Fiber cell. Figure modified from Kuszak & Brown, 1994. .......................................................................................................... 6 Figure 1.1.3: Overview of organelle degradation in the lens. Organelle degradation begins as lens epithelial cells enter the transition zone. Dnase2b becomes expressed, followed by nuclear lamina degradation. Lysosomes containing DNase2b fuse with the nuclease to drive DNA 3- cleavage. It generates 3’ PO4 DNA ends, which endogenous phosphatases convert to 3’-OH ends. The nucleus is then disintegrated and releases particulate material into the cytoplasm. Concurrently, Mitochondria are fragmented and organelles permeabilized, after which proteins are ubiquitinated, degraded, and cleared. Figure from Steven Bassnett, 2009. .......................................................................................... 9 vii Figure 1.2.1: Pax6 deficiency causes eye defects. Pax6 function