The polyadenosine RNA binding protein Nab2 regulates alternative splicing and intron retention during Drosophila melanogaster brain development

Abstract The regulation of cell-specific gene expression patterns during development requires the coordinated actions of hundreds of proteins, including transcription factors, processing enzymes, and many RNA binding proteins (RBPs). RBPs often become associated with a nascent transcript immediately after its production and are uniquely positioned to coordinate concurrent processing and quality control steps. Since RNA binding proteins can regulate multiple post-transcriptional processing steps for many mRNA transcripts, mutations within RBP-encoding genes often lead to pleiotropic effects that alter the physiology of multiple cell types. Thus, identifying the mRNA processing steps where an RBP functions and the effects of RBP loss on gene expression patterns can provide a better understanding of both tissue physiology and mechanisms of disease. In the current study, we have investigated the coordination of mRNA splicing and polyadenylation facilitated by the Drosophila RNA binding protein Nab2, an evolutionary conserved ortholog of human ZC3H14. ZC3H14 loss in human patients has previously been linked to alterations in nervous system function and disease. Both fly Nab2 and vertebrate ZC3H14 bind to polyadenosine RNA and have been implicated in the control of poly(A) tail length. Interestingly, we show that fly Nab2 functionally interacts with components of the spliceosome, suggesting that this family of RNA biding proteins may also regulate alternative splicing of mRNA transcripts. Using RNA-sequencing approaches, we show that Nab2 loss causes widespread changes in alternative splicing and intron retention. These changes in splicing cause alterations in the abundance of protein isoforms encoded by the affected transcripts and may contribute to phenotypes, such as decreases in viability and alterations in brain morphology, observed in Nab2 null flies. Overall, these studies highlight the importance of RNA binding proteins in the coordination of post-transcriptional gene expression regulation and potentially identify a class of proteins that can coordinate multiple processing events for specific mRNA transcripts. Author Summary Although most cells in a multicellular organism contain the same genetic material, each cell type produces a set of RNA molecules and proteins that allows it to perform specific functions. Protein production requires that a copy of the genetic information encoded in a cell’s DNA first be copied into RNA. Then the RNA is often processed to remove extra sequences and the finalized RNA can be used to create a particular type of protein. Our work is focused on how cells within developing fruit fly brain control the types, processing steps, and final sequences of the RNA molecules produced. We present data showing that when fly brain tissue lacks a protein called Nab2, some RNA molecules are not produced correctly. Nab2 loss causes extra sequences to be retained within many RNA molecules when those sequences are normally removed. These extra sequences can alter protein production from the affected RNAs and appear to contribute to the brain development problems observed in flies lacking Nab2. Since Nab2 performs very similar functions to a human protein called ZC3H14, these findings could provide a better understanding of how ZC3H14 loss leads to human disease.