The polyadenosine RNA-binding protein dNab2 interacts with the fragile X protein homolog and regulates gene expression in Drosophila neurons 公开
Bienkowski, Rick Stephen (2016)
Abstract
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
LIST OF FIGURES: 7
LIST OF TABLES: 8
CHAPTER 1: GENERAL INTRODUCTION 9
INTRODUCTION 10
I. RNA-BINDING PROTEINS ARE THE KEY MEDIATORS OF POST-TRANSCRIPTIONAL REGULATION IN EUKARYOTES 11
II. MUTATIONS IN THE ZC3H14 GENE CAUSE INTELLECTUAL DISABILITY 16
III. THE POLYADENOSINE RNA-BINDING PROTEIN DNAB2 IS THE DROSOPHILA MELANOGASTER HOMOLOG OF ZC3H14 23
IV. SUMMARY AND SCOPE OF THE DISSERTATION 28
FIGURES: 31
CHAPTER 2: THE DROSOPHILA ORTHOLOG OF THE ZC3H14 RNA BINDING PROTEIN ACTS WITHIN NEURONS TO PATTERN AXON PROJECTION IN THE DEVELOPING BRAIN 34
ABSTRACT 35
INTRODUCTION 36
MATERIALS AND METHODS 39
RESULTS 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 66
SUMMARY 67
INTRODUCTION 68
RESULTS 71
DISCUSSION 83
EXPERIMENTAL PROCEDURES: 87
FIGURES 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. 113
INTRODUCTION: 114
RESULTS: 115
CHAPTER 5: DISCUSSION AND CONCLUSION 125
I. SUMMARY: 126
II. A MODEL OF DNAB2 FUNCTION IN NEURONS 127
III. OPEN QUESTIONS AND FUTURE DIRECTIONS 128
III. CONCLUSION 140
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