The Disease-Associated Proteins Drosophila Nab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology Public
Rounds, John (Spring 2021)
Published
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 Nab2ex3 Adult Viability Through Angled Vials and the
Nab2Nex3F42A Outcross..................................................................................................................164
Results and Discussion..........................................................................................................................165
References............................................................................................................................................185
Figures.................................................................................................................................................188
CHAPTER 5: Discussion and Conclusion.........................................................................................204
Discussion............................................................................................................................................205
CONCLUSION.......................................................................................................................................218
References............................................................................................................................................220
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