University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 3-28-2019 Modeling Microcephaly Caused by Inactivation of the Minor Spliceosome Using a U11 Conditional Knockout Mouse Mary Baumgartner University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Baumgartner, Mary, "Modeling Microcephaly Caused by Inactivation of the Minor Spliceosome Using a U11 Conditional Knockout Mouse" (2019). Doctoral Dissertations. 2076. https://opencommons.uconn.edu/dissertations/2076 ABSTRACT Modeling Microcephaly Caused by Inactivation of the Minor Spliceosome Using a U11 Conditional Knockout Mouse Mary Baumgartner, Ph.D University of Connecticut, 2019 The minor spliceosome is one of two pre-mRNA splicing machineries required for protein-coding gene expression. In multiple eukaryotic lineages, the minor spliceosome is required to remove <1% of introns (minor introns) from the pre-mRNA transcripts of minor intron-containing genes (MIGs). Despite the few minor introns in the genome, disruption of minor splicing impairs development in numerous species. In humans, minor spliceosome mutations are linked numerous developmental diseases that impact central nervous system (CNS) development. However, the role of minor splicing and MIG expression in mammalian CNS development and function remains unexplored. To address this, I characterized the expression of the minor spliceosome-specific small nuclear RNA (snRNA) components and MIGs in the developing mouse embryo and retina. This approach revealed enriched expression of the minor spliceosome-specific snRNAs in the developing mouse CNS and limb buds, and in differentiating retinal neurons. I next sought to inactivate the minor spliceosome in the developing mouse cortex (pallium) by ablating Rnu11, which encodes the minor spliceosome-specific U11 snRNA. In the Rnu11 conditional knockout (cKO) pallium, U11-null radial glial cells (RGCs) displayed DNA damage, p53 accumulation, and cell cycle defects, culminating in self-amplifying RGC death and microcephaly. Conversely, neurons appeared to survive postnatally. These results suggested that MIG expression is essential for cycling cell survival, but is not immediately necessary for neuron survival. To investigate this, I leveraged publications identifying genes essential for cycling cell survival. MIGs were significantly enriched in the essential gene lists, underscoring their importance for cycling cell survival. In parallel, I explored how the developmental defects in the Rnu11 cKO mice affected their behavior in adulthood. The Rnu11 mutant mice displayed heightened anxiety and significant social and motor behavior impairments. Unexpectedly, Rnu11 heterozygous mice showed reduced sociability and enhanced whole-body motor performance. These findings suggest that U11 haploinsufficiency differentially impacts brain development/function, and identify the first phenotype associated with U11 haploinsufficiency. In all, these findings reveal the roles minor splicing plays in mammalian CNS development/function, laying the foundation for studies of disease pathogenesis and for research linking minor splicing to progenitor cell behavior, cell type- specific survival, and mouse behavior. i Modeling Microcephaly Caused by Inactivation of the Minor Spliceosome Using a U11 Conditional Knockout Mouse Mary Baumgartner B.A., Mount Holyoke College, 2013 M.S., University of Connecticut, 2016 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2019 ii APPROVAL PAGE Doctor of Philosophy Dissertation Modeling Microcephaly Caused by Inactivation of the Minor Spliceosome Using a U11 Conditional Knockout Mouse Presented by Mary Baumgartner, M.S. Major Advisor: ______________________________________________________ Rahul Kanadia, Ph.D Associate Advisor: ___________________________________________________ Joseph LoTurco, Ph.D Associate Advisor: ___________________________________________________ Jianjun Sun, Ph.D University of Connecticut 2019 iii Dedicated to my Grandmother, Barbara May Baumgartner, and my Grandfather, Reno Franconi iv ACKNOWLEDGEMENTS First, I would like to thank Dr. Rahul Kanadia for being a great mentor for the past six years. Not only did he dedicate countless hours guiding me to become better at writing, thinking, presenting, and interpreting science, but he always prioritized my training, bringing me a new opportunity almost every week. Without his presence to help push me into new scientific terrain, I would not be the scientist I am today. Second, I would like to thank my collaborator and director of the Murine Behavioral Neurogenetics Facility (MBNF) at the University of Connecticut, Dr. Holly Fitch, for sharing her expertise, facilities, and vast knowledge of mouse behavior, and for taking the time for our numerous meetings to discuss and interpret mouse behavioral data. I would also like to thank her students, Peter Perrino and Nicholas Buitrago, for performing the behavioral experiments at the MBNF. Third, I would like to thank my committee members, Dr. Joseph LoTurco and Dr. Jianjun Sun, for their unwavering support, advice, and invaluable suggestions during our meetings. They both were essential for helping me manage this at times unwieldy project. I would also like to thank the entire Kanadia lab, past and present, for their support and ability to make the most unrelenting day more bearable. Dr. Krishna Karunakaran, a past member of the lab and my first mentor, has always been an unending resource of advice, suggestions, and knowledge. I am not sure I could have survived my first SfN conference without her. I would also like to thank Dr. Abdul Rouf Banday, another past member of the lab, for his immense contributions and bioinformatics knowledge, which were both essential for the success of my first ever project in the lab. Similarly, Christopher Lemoine, another past member of the lab, was an amazing technical resource, and helped me troubleshoot many a protocol. I cannot imagine the countless hours he saved in the process. I would also like to thank Anouk Olthof, who is currently pursuing her PhD in the Kanadia lab, for her constant help and advice, both on scientific and non-scientific matters. Thank you to Anouk; Katery Hyatt, who is pursuing her Masters in the Kanadia lab; Alisa White, who is pursuing her PhD; Kyle Drake, who is pursuing his PhD; Dan Munteanu, who is pursuing his PhD; and Victor Calle, who is also pursuing his PhD, for always keeping the lab a positive place, despite how many experiments failed that day. Every one of you has always been available to help whoever might need it, either in the lab or outside of it, and you’ve all helped make the lab a true family. I would also like to thank the entire PNB department, who have always been available to provide advice or suggestions for any scientific question. In particular, I would like to thank the newly- minted Drs. Laura Mickelsen, for being the best roommate possible and for her support over the past five years, and Fu-Shan Kuo, for offering Laura and I a place to stay during a turbulent time and for being an incredible short-term roommate. I can’t thank them, and Fu-Shan’s husband Andres Barretto, enough for educating me on all the classic movies I somehow missed out on. I would also like to thank Drs. Geoffrey Tanner and Anastasios Tzingounis, for their invaluable career advice; Dr. Xinnian Chen, for opening my eyes to new methods of teaching and for the sheer amount of time she has dedicated over the years to making PNB 2274 as rewarding as possible; Dr. Kristen Kimball; Penny Dobbins; Ed Lechowicz; Ann Hamlin; and Sonda Davis for making PNB 2264/2274 both incredibly useful, in terms of experience, but also incredibly fun. I v would also like to thank Kathy Kelleher, Linda Armstrong, Noreen Sgro, and Taylor Renaud for their immense help navigating the administrative part of the program—especially for the last 6 months, which have been particularly rough. I would be lost without their help. Last but not least, I would like to thank my friends and family, who have been an incredible (and incredibly understanding) support system throughout this entire program, and for their constant encouragement whenever things were rocky, or when I would fall off of the face of the planet during long writing/research stretches. In particular, I’d like to thank my grandfather for his unwavering support, and for welcoming me into his house both for my first few months of graduate school and when I was commuting from Mount Holyoke College to spend a weekend working in the Kanadia lab. Speaking of emotional support, I would like to thank my cat-based support, for being both ridiculous and adorable: Khensu and Felix, along with Laura’s cats Addison (RIP), Roary, and Logan. Thank you! vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ......................................................................................................... v LIST OF FIGURES AND TABLES ............................................................................................ x Chapter 1: Minor intron splicing ................................................................................................ 1 1.1 RNA splicing ......................................................................................................................... 1 1.1.1 Intron definition: the concept