UNIVERSITY OF CALGARY Identification and Characterization of Murine Se70-2 by Stephanie E. Minnema A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY CALGARY, ALBERTA OCTOBER, 2006 © Stephanie E. Minnema 2006 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-26242-9 Our file Notre référence ISBN: 978-0-494-26242-9 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L’auteur conserve la propriété du droit d’auteur ownership and moral rights in this et des droits moraux qui protège cette thèse. Ni thesis. Neither the thesis nor la thèse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent être imprimés ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author’s permission. In compliance with the Canadian Conformément à la loi canadienne sur la Privacy Act some supporting forms protection de la vie privée, quelques may have been removed from this formulaires secondaires ont été enlevés de thesis. cette thèse. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n’y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Abstract Differentiation of embryonic stem (ES) cells enables the study of cellular differentiation in vitro. With an interest in regulatory genes in neural differentiation, the Rancourt lab established methods for in vitro neural differentiation of ES cells, and pursued a screen identifying genes expressed during this process. The Rancourt EST sequence dataset resulted; from this dataset the project described in this thesis arose. The work presented herein surrounds the identification and characterization of murine Se70-2 (mSe70-2), which is encoded by the gene Rbm26, represented in the Rancourt EST dataset as clone 3B6. Work presented includes the development and implementation of a data enrichment, management, and dissemination strategy for the Bain-Rancourt EST dataset: The Rancourt EST Database (RED). Analysis of the dataset by RED led to identification of 3B6, which adheres to criteria suggesting it may encode a nucleic acid binding protein involved in neural differentiation. The genomic structure of Rbm26 and a number of transcripts from the gene are identified. The analysis of Rbm26 mRNA expression in differentiating ES cell cultures, embryos, and adult tissues is shown. Expression of Rbm26 is higher in neural precursor enriched cell cultures than in spontaneously- or non-differentiating ES cells. Expression in embryos is greatest in neural and mesenchymal tissues and the timing of expression corresponds to the peak period of neurogenesis in the developing brain. Rbm26 encodes the murine homolog of human Se70-2, a putative nucleic acid binding protein. A paralog of mSe70-2 also exists, Psc1, which is a putative SR family splicing factor. Phylogenetic analysis indicates that mSe70-2 is conserved in eukaryotes, with gene duplication in vertebrate lineages. Characterization of mSe70-2 via antibody production and expression of the protein was pursued, but met with challenges. The possibility that mSe70-2 is regulated at the translational level is suggested. Knockdown of its C. elegans homolog indicates a possible role in positive regulation of the cell cycle, while the ability to bind RNA is shown using in vitro produced mSe70-2. Collectively, the results of this project suggest a role for mSe70-2 in cell fate decisions associated with differentiation along a subset of possible cell lineages. ii Acknowledgements Derrick Rancourt, for accepting me into your lab and being my academic father. Thank you also for allowing me to pursue my interests, even though they can be all over the place, figuratively and literally! I’ve learned an immense amount over the past years under your direction, and I know whichever direction I take in my career, I will be stronger with the skills you’ve given me. Doug Demetrick, for sitting on my committee. You have always been so approachable, and full of ideas and enthusiasm, even when it seemed the project was heading nowhere. I also want to thank you for the words of advice you gave me in 2005, when I thought my project was over: “fail now or fail later!” You gave me the perspective I needed to keep on going. Steve Robbins, for sitting on my committee despite your reservations about the project. Thanks you for all of your input into my project, and for helping me make sense of results that just don’t make sense. Christoph Sensen, for sitting on my committee and introducing me to Canadian Bioinformatics Workshops. I am indebted to you for the opportunities you’ve given me, and for your concern with my progress. I’ve really appreciated being able to drop in and chat (when you’re in town!) about science and life, and steal your never-ending supply of jelly beans. I’ll always remember the geese too! Torben Bech-Hansen, Angela Brooks-Wilson, Peter Bridge, and Jim McGhee, for taking time out of your busy schedules to contribute to my examinations or committee meetings during this process. Your time and interest are greatly appreciated… It also takes a village to raise a scientist! Rancourt Lab members, past and present. You guys have been my family for the past seven years. I’ve learned so much with you, not just about science, but also about life and myself. I’ll be taking with me so many memories when I go… the questionable lunch conversations, parties, window painting, joking around, Charles and Virginia the travelling mice, fighting over the music, trips to Lick’s, old Lloyd the drooling cat and Maggie’s weekend visits, creative pumpkin carving… iii Mike Wride and Fiona Mansergh, for establishing much of the work my project was built on. I miss you two; the lab just hasn’t been the same since you left! Shi-Ying Liu, for your endless efforts to get the 3B6 in situs working. I am genuinely touched by the care you put into helping me; I won’t forget your kindness. Eileen Rattner, for teaching me the finer points of ES cell culture. It’s an art form which you have mastered, and I feel lucky to have learned from the best. Ken Ito, the Zen master of molecular biology, and fellow mountain lover. I’ve learned so many little things from you… is there anything you don’t know? Jackie Irwin, fellow neat freak and mountain lover, for all of your assistance, great conversation, and organizational skills. It was a dark day when you left our lab; you were the only person who could keep on top of things! Knut Woltjen. We’re the last of the “original students” and it’s amazing we never tried to throw ourselves off the building. I could always count on you for an obscure conversation built on song lyrics, and great ideas from your molecular toolkit. Take care of Corkman, I’ll miss you both. Rebecca Everitt. Rebecca, I can’t even begin to thank you for everything you have contributed to this project, I could not have done it without you. You have taught me so much, and tolerated so many of my stupid questions. I envy your ability to stay calm even at the most stressful of times! I miss sharing an office with you, and I’ll miss the little visits with Alexandra and Trevor. Beattie Lab members, past and present. Thank you for welcoming me into your space and for the many reagents I borrowed. Your lab is a place of calm between the whirlwinds of the Rancourt labs, and I’m so lucky you guys moved in. I especially want to thank Tara, Brant, Deirdre, Chris, and Nick, for all of their excellent advice and ideas, which helped the progression of my work immensely. Jennifer Pelley, Sarah Attwell, Deirdre Lobb, and Maggie Renaud-Young, for critical reads of my thesis. You know you’ve got friends when they agree to read your thesis… I’m really thankful for your input! Ladies, you are fabulous! The friends I’ve met throughout the years. A most brilliant bunch, you are too many to name, but you know who you are. Graduate school: it’s the best of times; it’s the worst of times. I wouldn’t have made it through without you! You mean so much to me, iv even though some of you have long moved on from Calgary, and some of you are relative newcomers. Remember the times we’ve had… Fernie, Gary Bucy’s, Festivus, MSGSA kegs, martini parties, wine tasting, grad school complain-a-thons, Stampede BBQ, powder days, Robbie Burns, TGIF, Moose’s, Survivor watching, shakin’ it at the Embassy (or wherever), Halloween, science buddies, and on and on and on.