ZC3H14 is an evolutionarily conserved, ubiquitously expressed polyadenosine RNA-binding protein that is lost in an inherited form of non-syndromic intellectual disability (ID). Studies of ZC3H14 orthologs have revealed a conserved role for ZC3H14 in the restriction of poly(A) tail length, but the molecular function of this protein in neurons has not been defined. To further our understanding of ZC3H14 function in neurons we have utilized Drosophila melanogaster to model ZC3H14-associated ID. The Drosophila melanogaster ortholog of ZC3H14, dNab2, is required for viability in flies, and is critical for normal neuronal function and axon projection. Here we describe a network of physical and genetic interactions between dNab2 and the fragile X protein homolog dFMRP that link dNab2/ZC3H14 to translational repression. The dNab2 and dFMRP proteins co-precipitate from neurons and can be co-localized to cytoplasmic foci distributed along the neurites of cultured brain neurons. Two well-characterized dFMRP mRNA targets, futsch and CamKII, are repressed in a dNab2-dependent manner, providing strong evidence that dNab2 functions as a translational repressor in conjunction with dFMRP. In parallel, we find murine ZC3H14 enriched in axons of cultured primary hippocampal neurons and associated with the translational machinery, implying a conserved role for dNab2/ZC3H14 in the control of gene expression. These data suggest that dNab2/ZC3H14 contributes to dFMRP-mediated translational regulation of mRNAs trafficked to distal neuronal compartments, a process that is critical in neurons and may underlie brain-specific defects in ZC3H14-associated ID patients.
Table of Contents
Table of Contents
LIST OF FIGURES: 6
LIST OF TABLES: 7CHAPTER 1 : GENERAL INTRODUCTION 8 INTRODUCTION 9
I. RNA-BINDING PROTEINS ARE THE KEY MEDIATORS OF POST-TRANSCRIPTIONAL REGULATION IN EUKARYOTES 10
II. MUTATIONS IN THE ZC3H14 GENE CAUSE INTELLECTUAL DISABILITY 15
III. THE POLYADENOSINE RNA-BINDING PROTEIN DNAB2 IS THE DROSOPHILA MELANOGASTER HOMOLOG OF ZC3H14 22
IV. SUMMARY AND SCOPE OF THE DISSERTATION 27FIGURES: 29
CHAPTER 2 : THE DROSOPHILA ORTHOLOG OF THE ZC3H14 RNA BINDING PROTEIN ACTS WITHIN NEURONS TO PATTERN AXON PROJECTION IN THE DEVELOPING BRAIN 34ABSTRACT 35 INTRODUCTION 36
MATERIALS AND METHODS 39RESULTS 42 DISCUSSION 52 FIGURES: 55
CHAPTER 3: THE EVOLUTIONARILY CONSERVED RNA-BINDING PROTEIN DNAB2 INTERACTS WITH THE FRAGILE X PROTEIN HOMOLOG AND MEDIATES TRANSLATIONAL REPRESSION IN DROSOPHILA NEURONS 66SUMMARY 67 INTRODUCTION 68 RESULTS 72 DISCUSSION 83
EXPERIMENTAL PROCEDURES: 87FIGURES 93
CHAPTER 4 : THE POLYADENOSINE RNA-BINDING PROTEIN DNAB2 INTERACTS WITH THE RHO-GEF STILL LIFE TO PROMOTE VIABILITY AND PROPER DEVELOPMENT OF THE MUSHROOM BODIES IN DROSOPHILA MELANOGASTER. 120INTRODUCTION: 121 RESULTS: 122 CHAPTER 5 : DISCUSSION AND CONCLUSION 132
I. SUMMARY: 133
II. A MODEL OF DNAB2 FUNCTION IN NEURONS 134
III. OPEN QUESTIONS AND FUTURE DIRECTIONS 135
III. CONCLUSION 147List of Figures: Chapter 1:
Figure 1-1. The role of RNA-binding proteins (RBPs) in the maintenance of gene expression and in RNA metabolism.
Figure 1-2. Polyadenosine RNA-binding proteins (PABs) regulate mRNA expression
Figure 1-3. Domain structures of FMRP homologs are conserved between humansand Drosophila melanogaster.
Figure 1-4. Domain structure is conserved between human ZC3H14 and Drosophila melanogaster dNab2.Chapter 2:
Figure 2-1. dNab2 is required for proper development of the Drosophila mushroom body neurons.Figure 2-2. Loss of dNab2 disrupts the morphology of Drosophila mushroom body neurons.
Figure 2-3. dNab2 is expressed in the cell bodies of adult and pupa mushroom body neurons.
Figure 2-4. dNab2 is required in neurons for mushroom body development.
Figure 2-5. Mushroom body-specific expression of dNab2 rescues the Î²-lobemorphology defects in dNab2ex3 homozygous null mushroom bodies.
Figure 2-6. dNab2 is required for short-term memory.Chapter 3: Figure 3-1. Genetic interactions between dNab2 and dfmr1.
Figure 3-2. dNab2 and dfmr1 interact genetically in the process of mushroom body (MB) Î±-lobe development.
Figure 3-3. dNab2 localizes to neurites of primary brain neurons.
Figure 3-4. dNab2 physically associates with dFMRP in the neuronal cytoplasm.Figure 3-5. dNab2is required for translational suppression of futsch by exogenous dFMRP. Figure 3-6. dNab2 regulates expression of a CaMKII translational reporter.
Figure 3-7. ZC3H14 localizes to axons in primary hippocampal neurons and associates with polyribosomes in mouse cortical lysates.
Figure 3-S1: dFMRP regulates poly(A) tail length of the futsch mRNA transcript and of bulk RNA.Chapter 4: Figure 4-1. sif is the Drosophila melanogaster ortholog of human Tiam1 and Tiam2. Figure 4-2. Genetic interactions between sif and dNab2.
Figure 4-3. The sif mRNAtranscript is regulated by dNab2.
Figure 4-4. sif and dNab2 interact in the developing mushroom bodies.Chapter 5
Figure 5-1. A comprehensive model of dNab2 function in neurons.
Figure 5-2. Models of dNab2 binding specificity.List of Tables: Chapter 3:
Table 3-S1. Alleles tested for genetic interaction with dNab2 in a dNab2 overexpression eye screen.
About this Dissertation
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|The polyadenosine RNA-binding protein dNab2 interacts with the fragile X protein homolog and regulates gene expression in Drosophila neurons ()||2018-08-28||