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POST-TRANSCRIPTIONAL REGULATION of AFP and Igm GENES

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POST-TRANSCRIPTIONAL REGULATION of AFP and Igm GENES University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2011 POST-TRANSCRIPTIONAL REGULATION OF AFP AND IgM GENES Lilia M. Turcios University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Turcios, Lilia M., "POST-TRANSCRIPTIONAL REGULATION OF AFP AND IgM GENES" (2011). University of Kentucky Doctoral Dissertations. 210. https://uknowledge.uky.edu/gradschool_diss/210 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Lilia M. Turcios The Graduate School University of Kentucky 2011 POST-TRANSCRIPTIONAL REGULATION OF AFP AND IgM GENES ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Lilia M. Turcios Director: Dr. Martha Peterson Lexington, KY 2011 Copyright © Lilia M. Turcios 2011 ABSTRACT OF DISSERTATION POST-TRANSCRIPTIONAL REGULATION OF AFP AND IgM GENES Gene expression can be regulated at multiple steps once transcription is initiated. I have studied two different gene models, the α-Fetoprotein (AFP) and the immunoglobulin heavy chain (IgM) genes, to better understand post-transcriptional gene regulation mechanisms. The AFP gene is highly expressed during fetal liver development and dramatically repressed after birth. There is a mouse strain-specific difference between adult levels of AFP, with BALB/cJ mice expressing 10 to 20-fold higher levels compared to other mouse strains. BALB/cJ mice express low levels of Zhx2 and thus incompletely repress AFP. Despite differences in steady state AFP mRNA levels in the adult liver between Balb/cJ and wild-type mice, transcription rates across this gene were similar, indicating a post-transcriptional regulatory mechanism. I found accumulated unspliced RNA across multiple AFP introns in wild-type mice where mature AFP mRNA levels are low, suggesting overall AFP splicing is inefficient in the presence of Zhx2. The IgM gene is alternative processed to produce two mRNA isoforms through a competition between cleavage/polyadenylation (µspA) and splicing reactions and the pA/splice RNA expression ratio increases during B cell maturation. Cotranscriptional cleavage (CoTC) events, driven by specific cis-acting elements, are required downstream of some poly(A) signals to terminate transcription. In some cases, a pause site can produce similar effect. I explored whether there is a CoTC-like element within the IgM gene that may contribute to developmental changes in the mRNA ratio. In both a B cell and plasma cell line there was a gradual decrease in transcripts downstream from the µspA signal, suggesting that there is not evidence for a CoTC element within the IgM gene. To examine the effect a CoTC element would have on the competition between the splice and µspA reactions, we inserted the CoTC sequence of the β-globin gene into different locations downstream of the µspA signal. While the β- globin CoTC element caused cotranscriptional cleavage in all locations, it only affected the µspA/splice ratio when located close to the µspA site. This suggests there is a position effect of the inserted CoTC element on the competing polyadenylation and splicing reactions within the IgM transcripts. KEYWORDS: Postranscriptional regulation, Co-Transcriptional termination, RNA processing, IgM , AFP Lilia M. Turcios June 29, 2011 POST-TRANSCRIPTIONAL REGULATION OF AFP AND IgM GENES By Lilia M. Turcios Martha L. Peterson Director of Dissertation Beth Garvy Director of Graduate Studies June 29, 2011 RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor's degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date DISSERTATION Lilia M. Turcios The Graduate School University of Kentucky 2011 POST-TRANSCRIPTIONAL REGULATION OF AFP AND IgM GENES DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Medicine at the University of Kentucky By Lilia M. Turcios Director: Dr. Martha Peterson Lexington, KY 2011 Copyright © Lilia M. Turcios 2011 To Nicole and Samuel ACKNOWLEDGEMENTS I would like to thank Dr. Martha Peterson for giving me the opportunity to work under her supervision and for her remarkable support and patience in guiding me through the last five years, especially during the writing of this dissertation. I would also like to thank all of my dissertation committee members: Dr. Brett Spear, Dr. Charlotte Kaetzel and Dr. Brian Rymond for their valuable insights to improve the quality of this dissertation. I would like to extend my appreciation to the outside examiner, Dr. David Feola for his time and valuable comments as well to Dr. Qingjun Wang for sharing her expertise to optimize the Flag-tagged affinity purification. To Dr. Subbarao Bondada and Dr. Rebecca Dutch for allowing to perform rotations in their labs, which provided me a wonderful learning experience. I thank Dr. Jiyuan Ke for his guidance and confidence in me during my rotation in Dr. Bondada’s lab. I would like to express my gratitude to all the members of Dr. Spear’s lab for the enlightening discussions held during lab meetings and for the technical support especially from Erika Fleishaker, Michelle Glenn and Hui Ren who taught me how to deal with mice. To Amanda Ribble, an exceptional technician, for her invaluable assistance and patience during my first years in the lab and to Lorri Morford who initiated the Zhx2 project. To Jennifer Osterhage because her good sense of humor made the lab a more amenable place. I also extend my appreciation to all who helped me in anyway during my doctoral studies. I am thankful to the IBS program, The Graduate School and the Department of Microbiology, Immunology and Molecular Genetics for giving me the opportunity to pursue my doctoral studies at the University of Kentucky. I am grateful to Dr. Jane Harrison for her support and counseling during her time as Director of IBS program. I am thankful to my parents for their unconditional support and love and to my brothers, Carlos and Alfredo because I know they both will be there when I really need them. I am thankful to Andres for his exceptional support and his love for our children. And to Nicole and Samuel because they are the major motivation to reach the end and the reason for my existence. iii TABLE OF CONTENTS Acknowledgements ........................................................................................................ iii List of Tables ................................................................................................................. vii List of Figures ............................................................................................................... viii List of Abbreviations ........................................................................................................ x CHAPTER I: Introduction Introduction ..........................................................................................................1 Co-transcriptional RNA processing ...................................................................... 1 Splicing regulation ................................................................................................3 Co-transcriptional splicing and transcription coupling ........................................... 6 Alternative splicing and the nonsense-mediated decay pathway ......................... 7 Cleavage/polyadenylation and transcription termination by RNAPII ..................... 8 Alpha-fetoprotein function and developmental gene expression......................... 11 Zinc finger and homeoboxes (ZHX) family ......................................................... 14 AFP regulatory elements .................................................................................... 16 Regulation of AFP post-natal repression by zhx2 ............................................... 17 B Cell development ............................................................................................ 19 Regulation of IgM expression ............................................................................ 21 IgM transcription termination .............................................................................. 24 CHAPTER II: Materials and Methods Plasmid construction .......................................................................................... 30 Cell lines and culture conditions .................................................................... 31 B Cells lines ........................................................................................... 31
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