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A Trancriptomics Based Approach Reveals the Functional Consequences of Rnase MRP RNA Mutations in Yeast Md Shafiuddin a Disserta

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A Trancriptomics Based Approach Reveals the Functional Consequences of Rnase MRP RNA Mutations in Yeast Md Shafiuddin a Disserta A trancriptomics based approach reveals the functional consequences of RNase MRP RNA mutations in yeast. Item Type Dissertation Authors Shafiuddin, Md Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 27/09/2021 20:16:26 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/1802 A trancriptomics based approach reveals the functional consequences of RNase MRP RNA mutations in yeast Md Shafiuddin A Dissertation in Biochemistry and Molecular Biology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of State University of New York, Upstate Medical University Approved______________________ Date__________________________ i Acknowledgements There are many, without whom, this dissertation would not be possible. First and foremost, I would like to thank my thesis supervisor Dr. Mark Schmitt for allowing me to pursue my doctoral research in his lab. His outstanding support, guidance and patience throughout my time in graduate school were critical for my progress. I thank him for providing me hands-on training in numerous techniques in the lab and for sharing his knowledge and expertise in the field of RNase MRP biology. I would like to thank Dr. David Amberg and Dr. Xin Jie Chen for their valuable comments and suggestions as members of my thesis advisory committee. For me, one of the essential components for designing and performing experiments smoothly was the book “Methods in Yeast Genetics” and I thank Dr. Amberg for authoring such a useful book. I would like to thank Dr. Chen for his advice and encouragement during difficult periods of research. I appreciate his spending time with me discussing various aspects of mitochondrial biology. I have found his passion for science to be contagious. Next, I would like to thank present and past members of Schmitt lab – Dr. Qiaosheng Lu, Sara Wierzbicki, and Wayne Decatur. I would like to thank Wayne for all his help and advice, especially in matters related to bioinformatics. I have learned a lot from him and was lucky to have him as a mentor in the lab. I would like to thank my loving wife Fahima Sultana for all her support and companionship during all the ups and downs of this graduate program. Most important of all, I would like to thank my parents for their unconditional love and support throughout my life. Thank you all! ii Abstract A trancriptomics based approach reveals the functional consequences of RNase MRP RNA mutations in yeast By: Md Shafiuddin Sponsor: Dr. Mark E. Schmitt RNase MRP is a eukaryotic ribonucleoprotein complex involved in multiple cellular functions that includes ribosomal RNA processing, primer generation for mitochondrial DNA replication and degradation of cell cycle related mRNAs. In Saccharomyces cerevisiae, the RNA component of RNase MRP is encoded by NME1. We have performed random deletion mutagenesis of RNase MRP RNA gene and isolated a mutation, nme1-91, that causes temperature sensitive growth defect on glycerol media. RNA analysis of nme1-91 showed that this mutant is mildly deficient in the 5.8S rRNA processing function of RNase MRP. Growth analysis and northern blotting of RNase MRP RNA mutations generated based on nme1-91 allele suggested that 3’-end nucleotide sequences of the nme1-91 allele contribute to its phenotype. Highcopy suppression screen identified tRNA modification gene NCS6 as a suppressor of nme1-91. Additionally, primary mode of suppression by NCS6 was found to be non-mitochondrial since NCS6 partially suppressed the nme1-91 phenotype on fermentable carbon source. Strains carrying a deletion of NCS6 in combination with nme1-91 showed a synthetic sick phenotype. Polysome profile analysis of nme1-91 revealed that 80S monosomal fraction accumulates in this mutant. Differential gene expression analysis of nme1-91 by RNA- seq indicated that rRNA processing and cell cycle related genes become mis-regulated due to this mutation. A similar high-throughput sequencing based approach was also employed to investigate the transcriptional basis of positive genetic interactions between components of RNase MRP and nonsense-mediated decay pathway. A yeast strain bearing the nme1- P6 mutation in the RNA component of RNase MRP exhibits temperature-sensitive growth defect. This phenotype can be suppressed by deletion of NMD components. Differential gene expression analysis identified several mis-regulated biological processes in nme1-P6 and Δupf1 strains. Comparative transcriptomic analysis suggested that suppression of nme1-P6 phenotype by Δupf1 is accompanied by large shift in gene expression pattern towards Δupf1 strain. Moreover, the majority of direct targets of NMD were not down-regulated in nme1-P6 indicating that the effect of NMD on nme1-P6 might be due to increased degradation on mRNAs that are not targeted by NMD in normal conditions. Taken together, these results show that mutations of RNase MRP RNA can modulate diverse biological processes. iii Table of contents Title Page i Acknowledgements ii Abstract iii Table of contents iv List of Figures and Tables vi Chapter 1: Introduction 1 RNase MRP is a eukaryotic enzyme 2 Structural features of RNase MRP RNA 3 RNase MRP and RNase P are evolutionarily related enzymes 6 Cellular functions of RNase MRP 7 RNase MRP in human diseases 28 Nonsense Mediated Decay 33 Conclusion 35 Aims of research 36 References 38 Chapter 2: Molecular, genetic and transcriptomic analyses of an RNase MRP mutant that exhibits a mild deficiency in its 5.8S ribosomal RNA processing function 51 Abstract 52 Introduction 53 Materials and Methods 55 Results 62 Discussion 81 References 87 iv Chapter 3: A comparative transcriptomic analysis provides insight into the basis of a genetic interaction between RNase MRP and nonsense mediated decay components 92 Abstract 93 Introduction 94 Materials and Methods 98 Results 100 Discussion 113 References 118 Chapter 4: General discussion 125 Discussion 126 References 136 Appendix I: Mitochondrially directed MRP components do not interact with RNase MRP RNA inside mitochondria 139 Appendix II: A minimal RNase MRP RNA 146 Appendix III: Plasmids used in this study 151 Appendix IV: Supplementary data from Chapter 2 153 Appendix V: Supplementary data from Chapter 3 184 v List of Figures and Tables Chapter 1: Introduction Figure 1: Predicted secondary structure of Saccharomyces cerevisiae RNase MRP RNA 5 Figure 2: A schematic diagram showing the role of RNase MRP in mammalian mitochondrial DNA replication 9 Figure 3: Mitochondrial localization RMRP RNA in human 13 Figure 4: 5.8S maturation pathway in yeast Saccharomyces cerevesiae 20 Figure 5: Cellular functions of RNase MRP 27 Table 1: Genetic composition of RNase MRP and RNase P 3 Chapter 2: Molecular, genetic and transcriptomic analyses of an RNase MRP mutant that exhibits a temperature sensitive petite phenotype and a mild deficiency in its 5.8S ribosomal RNA processing function Figure 1: nme1-91 and its phenotypes 64 Figure 2: Growth phenotype of nme1-91 derived mutants 67 Figure 3: Genetic interactions between NME1 and NCS6 71 Figure 4: Network representations of mis-regulated processes in nme1-91 76 Figure 5: Alteration of polysome profiles due to nme1-91 mutation 81 Table 1: Saccharomyces cerevisiae strains used in this study 56 Table 2: Primers used in this study 58 Table 3: Top 5 enriched GO Cellular components in nme1-91 78 vi Chapter 3: A comparative transcriptomic analysis provides insight into the basis of a genetic interaction between RNase MRP and nonsense-mediated decay components Figure 1: Deletion of UPF1 partially suppresses nme1-P6 phenotype 103 Figure 2: nme1-P6 has a disomic chromosome IV 107 Figure 3: Venn diagrams showing the overlap between direct targets of NMD 110 Figure 4: Venn diagram showing the number of overlapping differentially expressed genes among strains 112 Figure 5: A probabilistic model for altered NMD activation in nme1-P6 117 Table 1: Saccharomyces cerevisiae strains used in this study 98 Table 2: Positive genetic interactions between MRP and NMD 101 Table 3: Top 3 GO biological processes mis-regulated in upf1Δ 105 Table 4: Top 3 GO biological processes mis-regulated in nme1-P6 105 Table 5: Top 3 GO biological processes up-regulated in upf1Δ according to Celik et al. 114 Appendix I: Mitochondrially directed MRP components do not interact with RNase MRP RNA inside mitochondria Figure 1: A schematic showing general architecture of MDMC constructs 140 Figure 2: Localization of mitochondrially directed MRP components 143 Figure 3: Immunoprecipitation of MDMCs 144 Appendix II: A minimal RNase MRP RNA Figure 1: A predicted secondary structure model for minimal MRP RNA 148 Figure 2: Growth analysis of a strain with minimal MRP RNA 149 vii CHAPTER 1 Introduction 1 RNase MRP is a eukaryotic enzyme Mitochondrial RNA processing ribonuclease (RNase MRP) is a eukaryotic enzyme with multiple cellular functions, most notable of which is the endonucleolytic cleavage of precursor ribosomal RNA (Chang and Clayton, 1987a; Schmitt and Clayton, 1993). In yeast Saccharomyces cerevisiae, this ribonucleoprotein complex is composed of a single RNA and 10 protein components. The RNA component is transcribed from the gene NME1 whereas protein constituents of RNase MRP are encoded by – POP1, POP3, POP4, POP5, POP6, POP7, POP8, RPP1, RMP1, and SNM1. Each of these 11 genes is essential in yeast, emphasizing the critical role of RNase MRP in eukaryotic life. RNase MRP is evolutionarily related to another RNA-protein complex named Ribonuclease P, or RNase P (Rosenblad et al., 2006; Woodhams et al., 2007). RNase P is a ribonuclease that endonucleolytically cleaves the 5'-leader sequence of pre-tRNAs and thus is an integral part of the tRNA maturation pathway (Jarrous and Gopalan, 2010). The universal conservation of RNase P in all life forms and the shared ubiquitous presence of RNase MRP and RNase P among all eukaryotes suggest that RNase MRP RNA and RNase P RNA diversed during early eukaryotic evolution.
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