The conserved RNA binding protein Nab2 regulates RNA targets critical for proper neuronal function Restricted; Files Only

Abstract

RNA binding proteins (RBPs) are critical for nearly every aspect of RNA processing and

regulation. These regulatory roles for RBPs are particularly important within highly

specialized cells such as neurons, which require enhanced fine-tuned spatiotemporal

regulation of gene expression to properly pattern complex neuronal networks within the

developing nervous system. One such RBP with critical roles in neurons and is required

for proper brain development and function is known as ZC3H14 (Zinc finger Cystine-

Cystine-Cystine-Histidine containing protein 14). ZC3H14 encodes a ubiquitously

expressed polyadenosine RBP with broad roles in post-transcriptional regulation of gene

expression. Moreover, loss of function mutations in human ZC3H14 cause a form of nonsyndromic

autosomal recessive intellectual disability (NS-ARID), however, our

understanding of ZC3H14-regulated RNAs as well as elevated roles for ZC3H14 in the

nervous system remain elusive. To investigate neurological functions of ZC3H14, we

employ Drosophila melanogaster as a model to explore the role of this conserved RBP in

neurodevelopment and neurodevelopmental disease. The Drosophila orthologue of

ZC3H14, known as Nab2 (Nuclear polyadenosine RNA-binding protein 2), is critical in

neurons and is required for proper brain development and function. Here, we present

RNA-sequencing analysis of Nab2null heads to define the effect of Nab2 loss on the

Drosophila head transcriptome. We further define a novel role for Nab2 in the regulation

of sex-specific splicing and m6A methylation status of the Drosophila Sxl transcript. We

then present evidence that Nab2 regulates splicing and abundance of Trio, a neuronallyenriched

Rho guanine nucleotide exchange factor (RhoGEF), to govern, in-part,

Drosophila brain morphology and neuronal architecture. In aggregate, these data suggest

that Nab2/ZC3H14 may function in neurodevelopment by regulation of m6A methylation

levels and splicing of select neuronally-enriched transcripts to regulate downstream

neurodevelopmental events such as regulation of m6A methylation, brain development,

and axon development. Taken together, these studies provide insight into human ZC3H14

function as well as ZC3H14-linked neurodevelopmental disease etiology.

Table of Contents

ABSTRACT ............................................................................................................................. vii

ACKNOWLEDGEMENTS .......................................................................................................... ix

CHAPTER 1: General IntroducDon ............................................................................................ 1

1.1 Regula*on of gene expression .................................................................................................... 2

1.1.a Regula*on of gene expression in the Nucleus ............................................................................................ 3

1.1.b Nucleocytoplasmic transport ..................................................................................................................... 9

1.1.c Regula*on of Gene Expression in the Cytoplasm ..................................................................................... 11

1.2 RNA binding proteins ............................................................................................................... 20

1.2.a RBPs in disease ......................................................................................................................................... 22

1.2.b Regula*on of RNA in the nervous system ................................................................................................ 23

1.3 Intellectual disability ................................................................................................................ 24

1.4 Drosophila melanogaster as a model for intellectual disability ................................................. 26

1.4.a The Drosophila Nervous System ............................................................................................................... 28

1.4.b Mushroom Bodies .................................................................................................................................... 29

1.4.c Dendri*c Arboriza*on Neurons ................................................................................................................ 31

1.5 ZC3H14-linked Intellectual Disability ........................................................................................ 32

1.5.a Mammalian ZC3H14 ................................................................................................................................. 32

1.5.b Caenorhabdi/s elegans Sut2 .................................................................................................................... 34

1.5.c Saccharomyces cerevisiae Nab2 ............................................................................................................... 34

1.5.d Drosophila melanogaster Nab2 ................................................................................................................ 37

1.6 Nab2 regulates splicing of select neuronally-enriched transcripts within the fly head. .............. 40

1.6.b Sex lethal (Sxl)- The master regulator of sex determina*on and dosage compensa*on in Drosophila ... 40

1.6.c The RhoGEF Trio ....................................................................................................................................... 43

1.7 Scope of Disserta*on ............................................................................................................... 46

1.8 Figures ..................................................................................................................................... 49

CHAPTER 2: The Drosophila Nab2 RNA binding protein inhibits m6A methylaDon and malespecific

splicing of Sex lethal transcript in female neuronal Dssue ......................................... 68

Abstract ......................................................................................................................................... 69

Introduc*on ................................................................................................................................... 70

Results ........................................................................................................................................... 72

Nab2 loss affects levels and processing of a subset of RNAs in the transcriptome of the Drosophila head ..... 72

Nab2 loss alters levels of transcripts linked to mRNA processing ..................................................................... 74

Nab2ex3 females exhibit masculinized Sxl splicing in neuron-enriched *ssues .................................................. 76

The dosage compensa*on complex contributes to phenotypes in Nab2ex3 mutant females ........................... 79

Loss of the MeYl3 m6A methyltransferase rescues Nab2ex3 phenotypes .......................................................... 80

Nab2 binds Sxl pre-mRNA and modulates m6A methyla*on ............................................................................. 82

Discussion ...................................................................................................................................... 87

Materials and Methods .................................................................................................................. 93

Acknowledgments ....................................................................................................................... 103

v

Compe*ng Interests ..................................................................................................................... 103

Figures ......................................................................................................................................... 104

CHAPTER 3: The RNA binding protein Nab2 regulates levels of the RhoGEF Trio to govern axon

and dendrite morphology ................................................................................................... 125

ABSTRACT .................................................................................................................................... 126

Significance Statement ................................................................................................................. 127

INTRODUCTION ........................................................................................................................... 128

RESULTS ....................................................................................................................................... 132

Loss of m6A-reader proteins rescues Nab2null defects in viability and adult locomo*on ................................ 132

Nab2 and MeYl3 regulate splicing of the trio 5’UTR in the Drosophila head ................................................. 133

Nab2 regulates levels of Trio M in the Drosophila head .................................................................................. 136

Trio is altered in the Nab2null mushroom body ................................................................................................ 137

Expression of Trio GEF2 rescues a/a’ and b’ defects in Nab2null mushroom bodies ....................................... 138

Expression of Trio GEF2 rescues Nab2null dendri*c arboriza*on defects in class IV ddaC sensory neurons ... 140

Expression of Trio GEF2 rescues Nab2null defects in viability and locomo*on ................................................. 140

DISCUSSION ................................................................................................................................. 141

METHODS .................................................................................................................................... 148

Acknowledgements ..................................................................................................................... 157

CHAPTER 4: EXPANDED DATA .............................................................................................. 171

Outcrossing the Nab2ex3 allele to improve Nab2ex3 adult viability ................................................. 171

Preliminary evidence suggests Sxl regulates mushroom body morphology in the female brain .... 173

Preliminary evidence suggests Nab2 causes ectopic methyla*on of the trio M transcript ............. 176

Nanopore direct sequencing of poly(A)+ RNA to map m6A modifica*ons within the Drosophila head

transcriptome .............................................................................................................................. 178

CHAPTER 5: DISCUSSION AND CONCLUSION ........................................................................ 184

APPENDIX: How to Select a Graduate School for a Ph.D. in Biomedical Science ................... 194

Abstract ....................................................................................................................................... 195

Author Biographies ...................................................................................................................... 196

Introduc*on ................................................................................................................................. 197

Ph.D. or Master’s? ....................................................................................................................... 198

Phase I: Research for Applica*ons ................................................................................................ 199

Phase II: The Interview ................................................................................................................. 210

Phase III: Follow-Up Research ...................................................................................................... 219

Acknowledgements ..................................................................................................................... 222

REFERENCES ........................................................................................................................ 224

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Molecular Biology, Genetics
Palabra Clave	neurodevelopment splicing RNA binding protein m6A
Committee Chair / Thesis Advisor	Anita Corbett, Emory University Leila Rieder, Emory University Ken Moberg, Emory University
Committee Members	James Zheng, Emory University David Gorkin, Emory University Gary Bassell, Emory University

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