The Disease-Associated ProteinsDrosophilaNab2 and Ataxin-2 Interact with Shared RNAs and Coregulate Neuronal Morphology

Abstract

Drosophila 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 Drosophila human ortholog ZC3H14 gives rise to an autosomal recessive intellectual disability, but understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part because no comprehensive identification of metazoan Nab2/ZC3H14-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships likely remain unidentified. Here we present evidence that Drosophila melanogaster 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 then present 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.

The RNA-binding protein Nab2 regulates the proteome of the developing Drosophila brain

“…The potential for this A-rich motif to be a Nab2 J o u r n a l P r e -p r o o f binding site is supported by the previous definition of a Nab2 binding motif in Saccharomyces cerevisiae (A11G and A12) (19,43,44). This Arich motif identified in the present study by examining conserved proteomics changes between Nab2 ex3 fly brains and ZC3H14 ∆ex13/∆ex13 mouse hippocampi is similar to a recently identified Arich motif defined via RNA-IP of fly Nab2 (39).…”

“…4 B , Nab2-labeled data point). Finally, the Nab2 ex3 differentially expressed and Nab2 oe differentially expressed datasets each have unique proteins that may provide insight into previously observed phenotypes in Nab2 mutants or overexpression systems ( 15 , 17 , 20 , 28 , 37 , 38 ). There are 152 proteins changed exclusively in Nab2 ex3 brains relative to control, and 311 proteins changed exclusively in the Nab2 oe brains relative to control ( Fig.…”