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Ribosomal Protein Mutants and Their Effects on Plant Growth and Development

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Ribosomal Protein Mutants and Their Effects on Plant Growth and Development RIBOSOMAL PROTEIN MUTANTS AND THEIR EFFECTS ON PLANT GROWTH AND DEVELOPMENT 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 Chad Dale Stewart © Copyright Chad Stewart, November, 2012. All rights reserved. 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 Ribosomes, large enzymatic complexes containing an RNA catalytic core, drive protein synthesis in all living organisms. 80S cytoplasmic eukaryotic ribosomes are comprised of four rRNAs and approximately 80 ribosomal proteins (r-proteins). R- proteins are encoded by gene families with large families (average of twelve members) predominating in mammals and smaller families (two to seven members) in plants. Increased ribosome heterogeneity is possible in plant ribosomes due to multiple transcriptionally active members in each family, whereas, in mammalian r-protein gene families, only one member is typically active. Multiple functional paralogs provide for greater plasticity in response to environmental/developmental cues, as well as, increasing the possibility of individual paralogs procuring or retaining extraribosomal functions. This research investigated the effects of r-protein mutations on plant growth and development. Through RNA interference (RNAi) mediated knockdown (KD) of type I (cytoplasmic: RPS15aA/D and F) and type II (non-cytosolic: RPS15aB and E) RPS15a family members I was able to confirm the delineation between the two types. Subcellular localization of the type I isoforms was nuclear/nucleolar while localization of type II isoforms was non- mitochondrial and probably cytosolic. Illumina sequencing of two r-protein mutant transcriptomes, pfl1 (rps18a) and pfl2 (rps13a), identified a novel set of up and down regulated genes, previously unknown or linked to r-protein mutants. The 20 genes identified were classified into four groups (1) plant defense, (2) transposable elements, (3) nitrogen metabolism and (4) genes with unknown function. Illumina miRNOME analysis revealed no changes in the miRNA profile of pfl1 and pfl2 plants. These data do not support the previously proposed theory that a disruption in ribosome biogenesis (by decreased r- protein synthesis) disrupts miRNA-mediated degradation of a range of auxin response genes. Finally, a novel double r-protein mutant, rps18a:HF/RPL18B, presented a late flowering/thickened bolt phenotype not seen in a rps13a:HF/RPL18B mutant, suggesting that RPS18A has an extraribosomal role in plant growth and development in Arabidopsis. ii ACKNOWLEDGEMENTS Without Dr. Peta Bonham-Smith’s constant encouragement and insight my time as a graduate student would not have been as enjoyable. She has proven to be an excellent mentor in not only my academic but also my personal life. Many thanks go to my committee members, Drs. Hong Wang, Chris Todd and Pierre Fobert for their numerous contributions to my project, and to Dr. Doug Muench for serving as my exteral examiner. Thank you also to Drs. Ken Wilson, Art Davis, Jose Andres and David Logan for access to both equipment and their expertise. I thank all past and present members of the Bonham-Smith lab for their friendship and assistance throughout my research including Drs. Rory Degenhardt, Donna Lindsay, Raghavendra Prasad Savada, Anoop Sindhu, Xianzhong Wu and Ushan Alahakoon, as well as, Heather Wakely, Esther McAleer, Alex Neumann, Marshall Timmermans, Jiangying Tu and Mitchell Baniulis. I would like to thank all members of the Biology Department, with special thanks to Marlynn Mierau, Jeaniene Smith, Joan Virgl, Diedre Wasyliw and Guosheng Liu. My deepest gratitude must be extended to my family and friends throughout this process, without you, I might have ended up in an Arts program. Special thanks to my parents Gord and Cheryl Stewart, Shauna Stewart, John Douglas, Jaret Laquerre, Dave Allan, Jodi Souter, Clare Anstead and Rebeccah Molnar. Lastly, I would like to acknowledge the financial contributions from the Natural Sciences and Engineering Research Council and the University of Saskatchewan College of Graduate Studies and Research. iii DEDICATION For their constant love, support and encouragement throughout this endeavor I dedicate this thesis to my wife Rebeccah Molnar and parents Gord and Cheryl Stewart. iv TABLE OF CONTENTS PERMISSION TO USE I ABSTRACT II ACKNOWLEDGEMENTS III DEDICATION IV TABLE OF CONTENTS V LIST OF TABLES VIII LIST OF FIGURES IX LIST OF ABBREVIATIONS XI 1. CHAPTER 1. LITERATURE REVIEW 1 1.1. Introduction 1 1.2. Ribosome structure and function 2 1.3. Ribosome assembly 3 1.3.1. Ribosome components 3 1.3.2. Ribosomal subunit biogenesis 3 1.3.3. Ribosomal subunit export 6 1.4. The nucleolus 6 1.5. Ribosomal proteins 8 1.5.1. Ribosomal protein functions 10 1.5.2. Ribosomal protein gene regulation 11 1.5.2.1. R-protein gene arrangement 11 1.5.2.2. Prokaryotic r-protein gene regulation 12 1.5.2.3. Eukaryotic r-protein gene regulation 13 1.5.2.3.1. Yeast r-protein gene regulation 13 1.5.2.3.2. Animal r-protein gene regulation 14 1.5.2.3.3. Plant r-protein gene regulation 15 1.6. Extraribosomal functions 16 1.7. Ribosomal protein mutants 19 1.8. Ribosomes and development 20 1.8.1. Ribosome deficiency model 20 1.8.2. Ribosome heterogeneity model 22 1.8.3. Ribosome aberrancy model 24 1.9. Arabidopsis thaliana RPS15a gene family 24 1.10. Objectives 25 v 2. CHAPTER 2. CHARACTERIZING THE RPS15A GENE FAMILY 27 2.1. Introduction 27 2.2. Material and methods 32 2.2.1. Plant material and growth conditions 32 2.2.2. RNAi constructs 32 2.2.3. Stable transgenics 32 2.2.4. Quantitative RT-PCR 33 2.2.5. Fluorescent protein constructs 34 2.2.6. RPS15aA/F and D C-terminal truncations 34 2.2.7. Transient expression in tobacco and confocal microscopy 35 2.2.8. RPS15aE overexpression in E. coli and western blots 36 2.2.9. Genevestigator transcript expression profiling 36 2.2.10. Statistics 37 2.3. Results 37 2.3.1. Type I and II RPS15a gene family members show similar expression patterns through a variety of developmental stages 37 2.3.2. Type I RPS15a isoforms exhibit classic r-protein subcellular localization while type II do not 39 2.3.3. Nuclear localization of the type I RPS15a isoforms requires the C-terminal seven amino acids of the protein 44 2.3.4. Delayed root development in individual type I and II RNAi lines 48 2.3.5. RPS15aE is not detected in total protein from Arabidopsis seedlings 56 2.4. Discussion 58 3. CHAPTER 3. USING NEXT GENERATION SEQUENCING TO ANALYZE pfl1 (rps18a) AND pfl2 (rps13a) RIBOSOMAL PROTEIN MUTANTS 67 3.1. Introduction 67 3.2. Material and methods 71 3.2.1. Plant material and growth conditions 71 3.2.2. Sample preparation for Illumina sequencing 71 3.2.3. Analysis of raw FASTQ data 72 3.2.3.1. Transcriptome analysis 72 3.2.3.2. miRNOME analysis 72 3.2.4. DESeq statistical analyses 73 3.2.5. Quantitative RT-PCR 73 3.2.6. Statistics 73 3.3. Results 73 3.3.1. Common changes in transcriptome in pfl1 and pfl2 compared to WT 73 3.3.2. qRT-PCR and Illumina data show similar trends of up and/or down regulation of Illumina identified transcripts 78 3.3.3. Transcript levels for Illumina-identified genes in tir1, afb1, afb2 and afb3 auxin mutants show little similarity to those in pfl1 and pfl2 78 3.3.4. miRNOME analysis of pfl1 and pfl2 showed no change in miRNA pools 84 3.4. Discussion 84 vi 4. CHAPTER 4. THE rps18a:HF/RPL18B DOUBLE MUTANT GENERATES A NOVEL PHENOTYPE 90 4.1. Introduction 90 4.2. Material and methods 94 4.2.1. Plant material and growth conditions 94 4.2.2. Fluorescent protein constructs 95 4.2.3. Transient expression in tobacco and confocal microscopy 95 4.2.4. Genevestigator transcript expression profiling 95 4.2.5. Light microscopy 95 4.3. Results 96 4.3.1. Heterozygous pfl1:HF/RPL18B double mutants result in a novel phenotype 96 4.3.2. Transcript expression profiles of each member of the RPS18, RPS13 and RPL18 families are consistent and similar across a variety of developmental stages 101 4.3.3. Nuclear and nucleolar subcellular localization of RPS13, RPS18 and RPL18 proteins 104 4.4. Discussion 110 5. CHAPTER 5. GENERAL DISCUSSION AND CONCLUSIONS 115 6. APPENDIX A. LIST OF R-PROTEINS IDENTIFIED IN 15-DAY-OLD ARABIDOPSIS TOTAL PROTEIN FOR CHAPTER 2 118 7.
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