The Drosophila RNA-binding Protein Nab2 Interacts with the Planar Cell Polarity Pathway to Regulate Neurodevelopment Öffentlichkeit

Abstract

RNA-binding proteins (RBPs) play critical roles in post-transcriptional regulation that have profound effects on gene expression. The importance of dysregulation of RNA processing is best illustrated by the numerous tissue-specific diseases associated with mutations in genes encoding RBPs, particularly neurological disorders. Neurodevelopment is highly complex and requires spatiotemporal control of gene expression, largely directed by RBPs. One such RBP is human ZC3H14, a ubiquitously expressed zinc-finger, polyadenosine RBP (ZnF CysCysCysHis #14), which has broad roles in post-transcriptional regulation, that is lost in an inherited form of intellectual disability. To gain insight into ZC3H14 neurological functions in the human brain, we employ the Drosophila model to explore the function of this evolutionarily conserved protein in neurodevelopment. Drosophila Nab2 (nuclear poly(A) binding protein 2), is the sole fly ortholog of ZC3H14. Here, we define roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies (MBs), which support olfactory learning and memory, and in regulating abundance of a small fraction of the total brain proteome, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Additionally, we show that components of the planar cell polarity (PCP) pathway are enriched in the Nab2 null brain proteome, that genetic data indicate Nab2 functions in guiding PCP-dependent MB axon projection and growth of larval sensory dendrites by a common, cell-autonomous mechanism, and finally reveal core PCP protein Van Gogh as a potential Nab2 target. In aggregate, these data demonstrate functions for Nab2 in axonal and dendritic development, provide a window into Nab2 regulated brain proteome, and identify interactions between Nab2 and components of the planar cell polarity pathway. These data suggest that Nab2/ZC3H14 may function in neurodevelopment through regulation of the PCP pathway by regulating trafficking and localization of PCP components, possibly identifying a mechanism underlying ZC3H14-linked intellectual disability in humans.

Table of Contents

Table of Contents:

Chapter 1: General introduction................................................................................................. 1

General Introduction.............................................................................................................. 2

1.1 Post-transcriptional gene regulation................................................................................. 3

1.1.a Nucleus................................................................................................................. 4

1.1.b Nucleocytoplasmic transport................................................................................ 6

1.1.c Cytoplasm............................................................................................................. 7

1.2 Importance of RBPs......................................................................................................... 9

1.2.a RBPs in mRNA processing.................................................................................. 9

1.2.b RBPs in disease.................................................................................................. 11

1.2.c Regulation of RNA in the brain......................................................................... 13

1.3 Drosophila as a model – of RBP function and disease.................................................. 15

1.3.a The Drosophila brain.......................................................................................... 15

1.3.b Mushroom Bodies.............................................................................................. 17

1.3.c Dendritic arborization neurons........................................................................... 18

1.4 Intellectual disability, ZC3H14, and Nab2.................................................................... 19

1.4.a Intellectual disabilities........................................................................................ 19

1.4.b Mammalian ZC3H14.......................................................................................... 20

1.4.c Drosophila Nab2................................................................................................. 21

1.4.d Planar cell polarity pathway based intellectual disability.................................. 22

1.5 Scope of dissertation...................................................................................................... 26

1.6 Figures............................................................................................................................ 28

Chapter 2: The RNA-binding protein Nab2 regulates the proteome of the developing Drosophila brain....................................................................................................................................................... 41

Abstract................................................................................................................................ 42

Introduction.......................................................................................................................... 43

Results.................................................................................................................................. 47

Discussion............................................................................................................................ 53

Experimental Procedures..................................................................................................... 58

References............................................................................................................................ 64

Figures................................................................................................................................ 106

Figure 2-1. Nab2 is required during pupal development for proper neuro-morphological patterning of the mushroom bodies............................................................................................ 107

Chapter 3: The RNA binding protein Nab2 patterns dendritic arbors and axons via the planar cell polarity pathway........................................................................................................................ 122

Summary............................................................................................................................ 123

Introduction........................................................................................................................ 124

Results................................................................................................................................ 129

Discussion.......................................................................................................................... 136

Experimental Procedures:.................................................................................................. 142

References.......................................................................................................................... 147

Figures................................................................................................................................ 186

Figure 3-5: Planar cell polarity components dominantly modify Nab2 dendritic phenotypes.  195

Chapter 4: Discussion and conclusions................................................................................... 209

4.1 Discussion.................................................................................................................... 210

4.1.a Neurodevelopment requires highly tuned post-transcriptional regulation....... 210

4.1.b Overview of main findings............................................................................... 210

4.2 Implications of Nab2 findings...................................................................................... 212

4.2.a Implications of Nab2 regulation of the brain proteome................................... 213

4.2.b Implications of Nab2 interaction with the PCP pathway................................. 218

4.2.c A model of Nab2 function in neurons.............................................................. 220

4.2.d Open questions and future directions............................................................... 221

4.3 Conclusions.................................................................................................................. 227

4.4 Figures.......................................................................................................................... 229

Chapter 5: References............................................................................................................... 233

 

List of Figures

Figure 1-1: Post-transcription gene regulation.............................................................................. 29

Figure 1-2: The Drosophila life cycle........................................................................................... 31

Figure 1-3: Drosophila mushroom body neuroanatomy............................................................... 33

Figure 1-4: Drosophila ddaC neuroanatomy................................................................................ 36

Figure 1-5: ZC3H14/Nab2 domain structure............................................................................... 38

Figure 1-6: Planar cell polarity pathway....................................................................................... 40

Figure 2-1. Nab2 is required during pupal development for proper neuro-morphological patterning of the mushroom bodies........................................................................................................................ 107

Figure 2-2. Study design and analytic approach for quantitative proteomic analysis of Drosophila pupal brains........................................................................................................................................... 109

Figure 2-3. Quantitative proteomic analysis of developmentally timed pupal brains reveals a role for Nab2 in neurodevelopment....................................................................................................................... 111

Figure 2-4. Nab2^ex3 and Nab2 oe brains display distinct sets of differentially expressed proteins but have similar changes among shared proteins....................................................................................... 113

Figure 2-5. Proteins increased in abundance in Nab2^ex3 and Nab2 oe brains are enriched for processes including RNA processing and neurodevelopment..................................................................... 115

Figure 2-6. Proteins reduced in abundance in Nab2^ex3 and Nab2 oe are enriched for neurological roles.    117

Figure 2-7. Six protein changes are shared between Nab2^ex3 flies and ZC3H14^{∆ex13/∆ex13} mice and contain a shared A-rich motif..................................................................................................................... 119

Figure 2-S1. A novel A-rich motif is shared among all transcripts corresponding to shared protein changes shared between Nab2^ex3 flies and ZC3H14^{∆ex13/∆ex13} mice.......................................................... 121

Figure 3-1: Nab2 loss alters levels of planar cell polarity pathway proteins in the Drosophila brain.        187

Figure 3-2: Planar cell polarity components dominantly modify Nab2 axonal phenotypes...... 189

Figure 3-3: Nab2 is required for proper dendritic development................................................. 191

Figure 3-4: Nab2 is required to restrict dendritic branching and projection............................... 193

Figure 3-5: Planar cell polarity components dominantly modify Nab2 dendritic phenotypes... 195

Figure 3-6: Nab2 is required for proper Vang localization in the central complex of the brain. 197

Figure 3-7: Zc3h14^∆13/∆13 mice have PCP-like cochlear defects................................................. 199

Figure 3-S1: Variance in mushroom body morphological defects with PCP modifying alleles. 201

Figure 3-S2: Proximal-distal effect on dendritic arbor complexity............................................ 203

Figure 3-S3: Overview of dominant modification of Nab2^ex3 phenotypes by PCP component alleles.        205

Figure 3-S4: Expanded view of Vang-eGFP localization........................................................... 207

Figure 4-1: A model of Nab2 function in neurons..................................................................... 230

Figure 4-2: Reynaud model of PCP mediated growth cone guidance.         232

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Neuroscience Biology, Cell
Stichwort	Drosophila
Committee Chair / Thesis Advisor	Anita H Corbett, Emory University Kenneth H Moberg, Emory University
Committee Members	Yue Feng, Emory University Dorothy Lerit, Emory University Gary Bassell, Emory University Tamara Caspary, Emory University

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