Cell-specific splicing factors that optimize calcium channel function Summer Elizabeth Allen B.A., Carleton College, 2005 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF NEUROSCIENCE AT BROWN UNIVERSITY PROVIDENCE, RHODE ISLAND MAY 2012 © 2010, 2012 Copyright by Summer Elizabeth Allen This dissertation by Summer Elizabeth Allen is accepted in its present form by the Department of Neuroscience as satisfying the dissertation requirement for the degree of Doctor of Philosophy. Date ____________________ ________________________________ Dr. Diane Lipscombe, Advisor Recommended to the Graduate Council Date ____________________ _______________________________ Dr. Barry Connors, Reader Date ____________________ ________________________________ Dr. William Fairbrother, Reader Date ____________________ ________________________________ Dr. Anne Hart, Reader Date ____________________ ________________________________ Dr. James Eberwine, Outside Reader Approved by the Graduate Council Date ____________________ ________________________________ Dr. Peter M. Weber, Dean of the Graduate School i Summer Elizabeth Allen Curriculum vitae Brown University T: 401-863-2615 Box GL-N, Sidney Frank Hall F: 401-863-1074 Department of Neuroscience [email protected] Providence, Rhode Island 02912 EDUCATION Brown University, Providence, Rhode Island 2005-present Ph.D. Candidate, Department of Neuroscience Dissertation title: Cell-specific splicing factors that optimize calcium channel function Advisor: Diane Lipscombe Carleton College, Northfield, Minnesota 2001-2005 B.A. in Biology, cum laude AWARDS AND FELLOWSHIPS Predoctoral Ruth L. Kirschstein National Research Service Award July 2009-Sept.2011 National Institute of Neurological Diseases and Stroke (F31NS066691) Title: "Finding factors that control cell-specific splicing of a calcium channel" Charles A. Dana Brain Science Interdisciplinary Fellowship Fall 2007 Brown University Institute for Brain Science RESEARCH EXPERIENCE Graduate Student 2005-present Brown University, Providence, Rhode Island Department of Neuroscience Dr. Diane Lipscombe, Advisor Investigating the proteins that regulate calcium channel alternative splicing. Research Assistant 2000-2004 Oregon Health and Sciences University, Portland, Oregon Department of Neuroscience Dr. M. Susan Smith and Dr. Kevin L. Grove Studied the neuronal circuitry involved in obesity. ii PUBLICATIONS In preparation Allen SE*, Phillips CG*, Raingo J, and Lipscombe D. (In preparation). The neuronal splicing factor Fox-2 controls Gs signaling to the N-type calcium channel. *SEA and CGP contributed equally to this work Published papers Allen SE, Darnell RB, Lipscombe D. 2010. The neuronal splicing factor Nova controls alternative splicing in N-type and P-type CaV2 calcium channels. Channels 4(6):483-9. Glavas MM, Grayson BE, Allen SE, Copp DR, Smith MS, Cowley MA, Grove KL. 2008. Characterization of brainstem peptide YY (PYY) neurons. J Comp Neurol. 10;506(2):194-210. Grayson BE, Allen SE, Billes SK, Williams SM, Smith MS, Grove KL. 2006. Prenatal development of hypothalamic neuropeptide systems in the nonhuman primate. Neuroscience. 28;143(4):975-86. Campbell RE, Smith MS, Grayson BE, Allen SE, ffrench-Mullen JMH, Grove KL. 2003. Y4 regulation of orexin neurons in the lateral hypothalamic area (LHA). J. Neurosci. 23:1487-1497. Grove KL, Allen S, Grayson BE, Smith MS. 2003. Postnatal development of the hypothalamic neuropeptide Y system. Neuroscience 116:393-406. Book chapter Lipscombe D, Allen SE, Gray AC, Marangoudakis S, Raingo J. 2008. Alternative splicing of neuronal CaV2 calcium channels. In: Kaczmarek L and Gribkoff VK (Eds.), Ion Channels. Wiley Publishers. P. 219-250. CONFERENCE ABSTRACTS Allen SE, Darnell RB, and Lipscombe D (2010) The neuronal splicing factor Nova-2 controls the expression of CaV2.1 and 2.2 splice isoforms. Society for Neuroscience. Phillips CG, Allen SE, Lipscombe D (2010) The neuronal splicing factor Fox-2 regulates an exon in Cav2.2 that controls the sensitivity of N-type calcium channels to inhibition by Gs proteins. Society for Neuroscience. Allen SE, Phillips CG, Lipscombe D (2010) The splicing factor Fox-2 controls N-type calcium channel activity in sympathetic neurons. Second Annual International Calcium Channel meeting. Phillips CG, Allen SE, Lipscombe D (2009). G protein-coupled receptor inhibition of N- type calcium channel splice-variants. Society for Neuroscience. Allen SE (2009). Finding factors that regulate the splicing of calcium channel exons. Brown-NIH Neuroscience Graduate Retreat at the Marine Biological Laboratory, Woods Hole, MA iii Allen SE, Lipscombe D. (2008). Fox proteins regulate the tissue-specific an development- specific alternative splicing of an N-type calcium channel exon. Society for Neuroscience. Allen SE, Xiao XQ, Grove KL, Smith MS. (2005). Neurokinin B (NKB) expression in the arcuate nucleus (ARH) is decreased during lactation. Endocrine Society Abstracts. TEACHING AND MENTORING Instructor July 2009 Psychopharmacology: Brain, body, and society Office of Continuing Education, Brown University -Developed and taught 15 hour, one week, pre-college course to 30 upper level high school students. -Designed syllabus, assignments, lectures, sheep brain dissection, and hands on activities; led discussions; wrote feedback to students. Graduate teaching assistant 2006 Neuroanatomy Department of Neuroscience, Brown University -Prepared for and conducted laboratory sessions, graded exams, and tutored undergraduate and graduate students. Mentor 2007-present Department of Neuroscience, Brown University -Teach experimental design, laboratory techniques, and data analysis to undergraduate student and graduate students. -Aid students in developing public speaking and writing skills. Professional development Harriet W. Sheridan Center for Teaching and Learning, Brown University Certificate I: Sheridan Teaching Seminar 2005-2009 Certificate II: Classroom Tools Seminar 2010 UNIVERSITY SERVICE Graduate Student Representative 2008-2009 Department of Neuroscience, Brown University - Planned graduate student recruitment weekends; served on admissions committee; acted as liaison between students and faculty. PROFESSIONAL AFFILIATIONS Society for Neuroscience 2005-present iv PREFACE Virtually all mammalian genes are alternatively spliced. The variation in protein isoforms caused by this alternative splicing is particularly prominent in the nervous system where the splicing of neuronal genes plays vital roles in axonal guidance, synapse differentiation, receptor modulation, and the regulation of neurotransmitter release. Cell-specific alternative splicing in voltage-gated calcium channels exemplifies how RNA processing can have dramatic effects on neuronal function. Calcium entry through presynaptic CaV2 channels initiates the intracellular cascade that triggers synaptic vesicle fusion and transmitter release, essential processes for neuronal communication. Tissue-specific splice isoforms of these channels have distinct basic biophysical properties and unique responses to G protein-coupled receptors. In my dissertation work I studied the factors that regulate tissue-specific splicing of CaV2 channels. Following introductory Chapter 1, in Chapter 2 I detail my discovery of a previously unreported alternative exon 24a in CaV2.1 mRNA. I also show that the neuronal-specific RNA-binding protein Nova-2 regulates inclusion of extracellular exons 24a and 31a in CaV2.1 and CaV2.2. In Chapter 3, I present our work exploring the function and regulation of alternative CaV2.2 exon 18a. Previous work from our lab showed that inclusion of this exon is developmentally regulated and depends on neuronal-cell type. In this thesis, I demonstrate that the splicing factor Fox-2 represses inclusion of CaV2.2 e18a. I also present work done in collaboration with Cecilia Phillips showing that inclusion of e18a in sympathetic neurons renders CaV2.2 channels uniquely susceptible to Gs-mediated voltage-independent inhibition. The studies presented in v Chapter 3 are unique because they show how a specific alternative exon regulated by a cell-specific splicing factor protein controls a fundamental neuronal process. In Chapter 4, I present my attempts to identify the factors that regulate the splicing of mutually exclusive exons 37a and 37b in CaV2.2. Splicing of these exons is both tissue-specific and functionally important. Inclusion of exon 37a in CaV2.2 channels in neurons of dorsal root ganglia allows Gi/o coupled-receptor agonists to inhibit CaV2.2 channels in a voltage- independent manner. Although I have yet to identify the splicing factors involved, I present evidence from minigene studies that suggests a model that includes a repressor of exon 37a and an enhancer of exon 37b. Bioinformatic analyses suggest that hnRNP- A/B and hnRNP-F may regulate inclusion of these exons. In the discussion chapter I examine how studies such as mine can provide important insight into cell-specific optimization of protein function. I also discuss models of splicing regulation and exciting avenues for future research. vi ACKNOWLEDGMENTS First and foremost, I would like to acknowledge my advisor, Diane Lipscombe. Diane has been an amazing mentor. She is able to tailor her mentorship style to fit each particular mentee. For me that’s meant that she has given me freedom to explore new ideas and techniques while offering guidance and encouragement when needed. She’s helped me