Box C/D snoRNP assembly factors regulate translation and contribute to disease Restricted; Files Only

Dreggors-Walker, Liz (Summer 2022)

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Regulation of protein synthesis is critical for the control of gene expression in all cells. Ribosomes are RNA-protein machines responsible for translating all proteins, and defects in ribosome production, function, or regulation result in disease. Ribosomal RNA (rRNA) is highly modified, and these modifications are critical for proper ribosome biogenesis and translation. One abundant class of rRNA modifications is 2’-O-methylation. 2’-O-methylations are guided by box C/D small nucleolar RNAs (snoRNAs), which assemble with evolutionarily conserved proteins to form active RNA-protein complexes (snoRNPs) to deposit rRNA modifications. The assembly of snoRNPs relies on several transient assembly factors through a poorly understood mechanism. In this dissertation, I use budding yeast as a model system to uncover a novel mechanism in snoRNP assembly, novel aspects of snoRNA-mediated regulation of translation, and how dysregulation of snoRNP assembly can lead to disease. The first part of this dissertation characterizes the box C/D snoRNP assembly factor Bcd1. We show that interactions between Bcd1 and Snu13 as well as interactions between Bcd1 and snoRNAs are critical for snoRNP assembly, unveiling a novel step in the hierarchal assembly of snoRNPs. Next, we use a mutant with impaired Bcd1 function and decreased rRNA 2’-O-methylation to determine how rRNA 2’-O-methylations impact translation. We reveal that hypo 2’-O-methylation impacts ribosome efficiency and fidelity by altering ribosome dynamics and the rotational status of ribosomes. The bulk of my dissertation work has focused on characterizing the box C/D snoRNP assembly factor Hit1. Mutations in the human homolog of Hit1 (ZNHIT3) result in the devastating neurodevelopmental disease PEHO syndrome. I generated budding yeast models of these PEHO-linked variants. My studies revealed that the PEHO-linked mutants in yeast result in loss of box C/D snoRNAs, impaired rRNA processing, decreased levels of rRNA 2’-O-methylations, impaired translation fidelity, and impaired ribosomal ligand binding. These data provide the first insights into the molecular basis of PEHO syndrome and suggest that PEHO syndrome is a ribosomopathy that is likely caused by impaired translation. Taken together, the studies presented in this dissertation reveal novel aspects of box C/D snoRNP biogenesis that are essential for proper translation and suggest that dysregulation of snoRNP assembly can result in human disease. 

Table of Contents

Table of Contents

Chapter 1. Introduction: insights into the regulation of ribosome biogenesis by ribosomal RNA modification 1

1.1 Small nucleolar RNAs and their cellular functions 1

1.2 Processing of snoRNAs and snoRNP assembly 4

1.3 The impact of snoRNAs on ribosome biogenesis in yeast 6

1.4 Methods for the study of snoRNAs in yeast 11

1.5 Summary of introduction 16

1.6 References 18

1.7 Figures 34

Figure 1. Box C/D and box H/ACA snoRNAs contain conserved sequence features and bind evolutionarily conserved proteins to form active snoRNPs. 34

Figure 2. Secondary structure of the yeast 18S rRNA highlighting snoRNA and protein-binding regions. 35

Figure 3. Secondary structures of the yeast 25S rRNA highlighting snoRNA and protein-binding regions. 37

1.8 Tables 40

       Table 1. Tools and methods to explore snoRNA function 40

Chapter 2. Studies in a budding yeast model suggest ribosomal defects as the molecular basis for PEHO syndrome 41

2.1 Summary 41

2.1 Abstract 42

            2.2 Introduction 42

            2.3 Materials and Methods 45

            2.4 Results 49

            2.5 Discussion 56

            2.6 Acknowledgements 61

            2.7 References 62

            2.8 Figures 72

Figure 1. PEHO syndrome variations in the evolutionarily conserved protein Hit1. 72

Figure 2. PEHO syndrome-associated amino acid variations in Hit1 protein cause temperature-sensitive slow-growth phenotypes and result in significant loss of Hit1 protein. 73

Figure 3. PEHO syndrome mutations result in defective rRNA processing and a reduction of steady-state box C/D snoRNA levels. 75

Figure 4. PEHO syndrome mutations cause translation defects. 77

Figure 5. The hit1-C11F mutation results in site-specific reductions of 2’-O-methylation levels of polysomal rRNAs. 79

Figure 6. The hit1-C11F mutant impacts 2′-O-methylation of nucleotides at key ribosome positions. 81

Figure 7. Model of the molecular mechanism of cellular defects caused by PEHO syndrome-associated Hit1 variants. 83

Figure S1. HA-tagged HIT1 mutant yeast have the same slow-growth phenotype as untagged HIT1 mutant yeast. 84

Figure S2. Ribosomal RNA 2’-O-methylation is not significantly altered between the total and polysomal RNA pools. 85

Figure S3. Views of the small ribosomal subunit. 86

Chapter 3. A conserved Bcd1 interaction essential for box C/D snoRNP biogenesis 87

            3.1 Summary 87

3.2 Abstract 88

            3.3 Introduction 88

            3.4 Materials and methods 90

            3.5 Results 95

            3.6 Discussion 102

            3.7 Acknowledgements 106

            3.8 References 107

            3.9 Figures 116

Figure 1. A conserved residue in the N-terminal region of Bcd1 is important for cell growth. 116

Figure 2. In vitro protein-binding assays testing the interaction network of Bcd1 with its binding partners. 118

Figure 3. Mutation of a conserved residue of Bcd1 hampers the ability of the protein to interact with RNA. 119

Figure 4. Bcd1-D72A variant abrogates the production of box C/D snoRNAs. 121

                        Figure 5. Ribosome biogenesis is impaired in cells expressing Bcd1-D72A. 122

                        Figure 6. Alteration of rRNA processing in Bcd1-D72A cells. 123

Figure S1. Sequence alignment of the fragments of Bcd1 corresponding to S.cerevisiae residues 47-97 from different organisms. 125

                        Figure S2. Pull downs. 126

                        Figure S3. Analyses of ZNHIT6 cancer mutations. 127

Chapter 4. Ribosomal RNA 2'- O-methylations regulate translation by impacting ribosome dynamics 128

            4.1 Summary 128

4.2 Abstract 129

            4.3 Introduction 129

            4.4 Materials and methods 132

            4.5 Results 136

            4.6 Discussion 147

            4.7 Acknowledgements 152

            4.8 References 153

            4.9 Figures 163

Figure 1. rRNA 2’-O-methylation sites change in a site-specific manner in bcd1-D72A cells. 163

Figure 2. rRNA hypomethylation affects the function and fidelity of ribosomes. 164

Figure 3. Hypo 2’-O-methylated ribosomes adopt a more rotated conformation in vivo. 166

Figure 4. Binding of eIF1 to hypomethylated 40S is weakened in vivo and in vitro. 168

Figure S1. Comparison of the 2’-O-methylation sites. 170

Figure S2. Comparing the modification and composition of mature ribosomes from BCD1 and bcd1-D72A cells. 171

Figure S3. nop1-ts affects the rRNA 2’-O methylation but not the snoRNA levels. 173

Figure S4. Position of rRNA 2’-O-methylations relative to eIf1 and the E- and P-site tRNAs.  175

Figure S5. Comparing the position of SSU-A579 in the open and closed conformations. 176

Figure S6. Comparison of the changes in rRNA 2’-O-methylation levels of the sites conserved between yeast and human. 177

Chapter 5. Discussion 178

5.1 Summary 178

5.2 Implications for understanding the fundamental aspects of snoRNP assembly and translation regulation 179

5.3 Implications for disease 180

5.4 Future directions to uncover the role of assembly factors for snoRNP assembly 182

5.5 Future directions to reveal the role of rRNA modifications in translation 183

5.6 Future directions for the study of PEHO syndrome 185

5.7 References 188

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