The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology Público

Abstract

Nab2 encodes a conserved polyadenosine RNA-binding protein (RBP) with broad roles in post-transcriptional regulation, including in poly(A) RNA export, poly(A) tail length control, transcription termination, and mRNA splicing. Mutation of the Nab2 human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability, but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited. No comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted, and many Nab2/ZC3H14 functional protein partnerships likely remain unidentified. Moreover, the global effects of Nab2/ZC3H14 loss on RNAs and proteins are either incompletely understood or, more often, unknown. Here we present an RNA-sequencing experiment defining the effects of Drosophila melanogaster Nab2 loss on RNA abundance and structure in neuron-enriched head tissue at high resolution. We then present evidence that Drosophila Nab2 interacts with the RBP Ataxin-2 (Atx2), a neuronal translational regulator, and implicate these proteins in coordinate regulation of neuronal morphology and adult viability. We next detail the first high-throughput identifications of Nab2- and Atx2-associated RNAs in Drosophila brain neurons using an RNA immunoprecipitation-sequencing (RIP-Seq) approach. Critically, the RNA interactomes of each RBP overlap, and Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with a small fraction of all polyadenylated RNAs. The identities of shared associated transcripts (e.g. drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g. Arpc2, tea, respectively) promise insight into neuronal functions of and interactions between each RBP. Significantly, Nab2-associated RNAs are overrepresented for internal A-rich motifs, suggesting these sequences may partially mediate Nab2 target selection. Taken together, these data demonstrate that Nab2 opposingly regulates neuronal morphology and shares associated neuronal RNAs with Atx2, and that Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest. By identifying RNAs associated with neuronal Nab2, our results supply clear direction for the ongoing study of Drosophila Nab2, enabling future definition of the precise molecular function of this RBP on its neuronal target RNAs. Beyond these insights, our results provide an essential foundation for expanding understanding of human ZC3H14 and ZC3H14-linked intellectual disability and neuronal development.

Table of Contents

List of Figures...........................................................................................................................................i

List of Tables...........................................................................................................................................ii

CHAPTER 1: General Introduction....................................................................................................1

INTRODUCTION......................................................................................................................................2

References.............................................................................................................................................12

CHAPTER 2: The Nab2 RNA binding protein promotes sex-specific splicing of Sex lethal in Drosophila

neuronal tissue..............................................................................................................................18

ABSTRACT..............................................................................................................................................19

Introduction...........................................................................................................................................20

Results...................................................................................................................................................23

Discussion..............................................................................................................................................34

Materials and Methods............................................................................................................................38

Acknowledgments...................................................................................................................................47

References..............................................................................................................................................48

Figures and Tables...................................................................................................................................54

CHAPTER 3: The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs

and Coregulate Neuronal Morphology..............................................................................................76

ABSTRACT..............................................................................................................................................77

Introduction...........................................................................................................................................78

Materials and Methods............................................................................................................................82

Results...................................................................................................................................................94

Discussion............................................................................................................................................105

Acknowledgements...............................................................................................................................114

Literature Cited....................................................................................................................................116

Figures and Tables................................................................................................................................128

Supplemental Materials and Methods.....................................................................................................158

Custom Software Code...........................................................................................................................163

CHAPTER 4: Expanded Data | Other Tests of Nab2-Atx2 Interaction, RIP-Seq rRNA Depletion and Read

Depth Limitations, and Improvements in Nab2^ex3 Adult Viability Through Angled Vials and the

Nab2^Nex3F42A Outcross..................................................................................................................164

Results and Discussion..........................................................................................................................165

References............................................................................................................................................185

Figures.................................................................................................................................................188

CHAPTER 5: Discussion and Conclusion.........................................................................................204

Discussion............................................................................................................................................205

CONCLUSION.......................................................................................................................................218

References............................................................................................................................................220

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, Bioinformatics Biology, Genetics Biology, Molecular
Palavra-chave	mushroom body Nab2 intellectual disability RNA-sequencing (RNA-Seq) ZC3H14 polyadenosine RNA-binding RNA immunoprecipitation-sequencing (RIP-Seq) RNA-binding proteins Drosophila associated RNAs Atx2
Committee Chair / Thesis Advisor	Anita H. Corbett, Emory University Kenneth H. Moberg, Emory University
Committee Members	Roger Deal, Emory University James Zheng, Emory University Gary Bassell, Emory University Yue Feng, Emory University

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