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CHARACTERIZATION of REGULATION of EXPRESSION and NUCLEAR/NUCLEOLAR LOCALIZATION of ARABIDOPSIS RIBSOMAL PROTEINS a Thesis Submit CHARACTERIZATION OF REGULATION OF EXPRESSION AND NUCLEAR/NUCLEOLAR LOCALIZATION OF ARABIDOPSIS RIBSOMAL PROTEINS A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Biology University of Saskatchewan Saskatoon By Raghavendra Prasad Savada Copyright Raghavendra Prasad Savada, June, 2011. All rights reserved. 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: 978-0-494-92118-0 Our file Notre référence ISBN: 978-0-494-92118-0 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, distrbute 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 protege 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. PERMISSION TO USE In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Biology University of Saskatchewan Saskatoon, Saskatchewan S7N 5E2 i ABSTRACT Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes. ii ACKNOWLEDGEMENTS First, I would like to thank my supervisor Dr. Peta Bonham-Smith for her support and encouragement throughout my PhD. I would like to thank my advisory committee members Drs. Chris Todd, Jonathan Page, and Pat Krone for all their valuable suggestions and feedback. I would also like to thank Drs. Ken Wilson, David Logan, Susan Kaminskyj, and Neil Chilton for allowing me to use their lab equipment and providing expertise. Thank you very much Drs. Rory Degenhardt, Kerri McIntosh, Jacqueline Hulm, Donna Lindsay, Anoop Sindhu, Ms. Heather Wakely and Mr. Chad Stewart for all your help and friendship. I would also like to thank undergraduate students of BIOL420 (2009-2010) for their technical assistance. I would like to thank the faculty, staff (especially Bonita Wong, Joan Virgl and Deidre Wasyliw) and graduate students of the Department of Biology for guiding me through my program. My sincere thanks to Dr. Gousheng Liu and Marlynn Mierau for all the technical assistance you provided me. I acknowledge the financial support of University of Saskatchewan for my program. iii TABLE OF CONTENTS PERMISSION TO USE I ABSTRACT II ACKNOWLEDGEMENTS III TABLE OF CONTENTS IV LIST OF TABLES VII LIST OF FIGURES VIII LIST OF ABBREVIATIONS IX CHAPTER I. LITERATURE REVIEW 1 1.1. Introduction 1 1.2. The ribosome, a two subunit ribozyme complex 2 1.2.1. rRNAs and their functions 3 1.2.2. Ribosomal proteins (RPs) 4 1.2.2.1. Features of RPs 4 1.2.2.2. Conservation of RPs 5 1.2.2.3. Functions of RPs 6 1.2.2.3.1. Ribosome biogenesis and translation 6 1.2.2.3.2. Selective translation of mRNAs 7 1.2.2.3.3. Extraribosomal functions 8 1.2.2.3.3.1. Regulation of gene expression 8 1.2.2.3.3.2. Enzymes 9 1.2.2.3.3.3. Regulation of cell proliferation, apoptosis and tumorigenesis 10 1.2.2.3.3.4. Plant growth, development, biotic and abiotic stress 10 1.2.2.4. RP Gene expression 12 1.2.2.4.1. Transcription 12 1.2.2.4.2. Post-transcription and translation 13 1.2.2.5. Coordinated regulation of RP gene expression 14 1.3. The nucleolus – the site of ribosomal subunit biogenesis 16 1.4. Nuclear localization of proteins 18 1.5. Arabidopsis ribosomal protein gene family RPL23a 21 1.6. Objectives 22 CHAPTER 2. CHARACTERIZATION OF NUCLEAR/NUCLEOLAR LOCALIZATION OF ARABIDOPSIS RIBOSOMAL PROTEINS RPL23AA, RPL15A AND RPS8A 23 iv 2.1. Introduction 23 2.2. Materials and methods 27 2.2.1. Plant material 27 2.2.2. Site-directed mutagenesis 27 2.2.3. Fluorescent protein fusion constructs 29 2.2.4. Transient expression in tobacco and confocal microscopy 30 2.2.5. Yeast two hybrid assay 30 2.3. Results 31 2.3.1. Mutation of pNLS 10KKADPKAKALK20 in RPL23aA did not affect nuclear or nucleolar localization 31 2.3.2. Individual pNLS mutations had no effect on localization, while simultaneous mutations did affect nucleolar localization of RPL23aA 31 2.3.3. Simultaneous mutation of pNoLS2, 5, 6 and 3 did not affect nucleolar localization, but serial mutation of the remaining pNoLSs increasingly disrupted nucleolar localization 36 2.3.4. Nucleolar localization of RPL23aA requires a combined number of pNoLSs, rather than any specific pNoLSs 36 2.3.5. The N-terminus is dispensable, while the C-terminus is required for nucleolar localization of RPL23aA 41 2.3.6. Mutation of pNoLSs increased nuclear and cytoplasmic intensity of RPL23aA- EGFP fusions 44 2.3.7. RPL23aA did not interact with any of the importin αs 44 2.3.8. Signal requirements for nuclear/nucleolar localization of RPL15A and RPS8A differ to those required for RPL23aA 44 2.4. Discussion 55 CHAPTER 3. DIFFERENTIAL REGULATION OF EXPRESSION AND SUBCELLULAR LOCALIZATION OF ARABIDOPSIS RIBOSOMAL PROTEINS 62 3.1.
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