The post-transcriptional regulation of RNA is crucial for establishing the gene expression profile of individual cells. Strict regulation of post-transcriptional events is important in neurons, as the spatial and temporal regulation of protein synthesis is critical for proper neural development. The addition of a poly(A) tail to the 3'- end of mRNA transcripts is a key post-transcriptional event that influences the export, stability, transport and translation of transcripts. Our lab has recently found that mutations in the ZC3H14 gene, which encodes an evolutionarily conserved zinc finger RNA-binding protein, cause an autosomal recessive non-syndromic form of intellectual disability. ZC3H14 binds polyadenosine RNA with high affinity in vitro and loss of ZC3H14 leads to extended poly(A) tails in a subset of RNAs. However, the function of ZC3H14 in neurons and the underlying brain dysfunction in intellectual disability patients is unknown. To address this gap in knowledge, we have developed a fly model of ZC3H14-associated intellectual disability by creating a null allele of the Drosophila melanogaster ortholog of ZC3H14, dNab2. In flies, loss of dNab2 causes extended poly(A) tails along with a number of neuronal-specific and neurodevelopmental phenotypes. Importantly, these neuronal defects can be rescued by expression of human ZC3H14, validating the use of the fly model to investigate the role of ZC3H14. The long-term goal of my studies is to understand how polyadenylation is regulated by ZC3H14 family RNA binding proteins in order to provide insights into the molecular defects that underlie brain dysfunction in ZC3H14-associated intellectual disability. Based on intriguing preliminary data, including genetic and physical links to the Drosophila counterpart of the Fragile X Mental Retardation protein (FMRP/dFMR1), we will test the hypothesis that dNab2 regulates a set of RNAs that are critical for proper neurodevelopment and function, a subset of which are also bound and co-regulated by dFMR1. This hypothesis will be tested though two complementary aims: 1) Identify specific RNA targets of dNab2 in neurons using an optimized Crosslinking RNA immunoprecipitation (RNA-CLIP) approach and examine how loss of dNab2 impacts these transcripts thus providing insight into the function of dNab2/ZC3H14, and 2) Further characterize an interaction that we have identified between dNab2 and dFMR1 in neurons to provide insight into how dNab2 and dFMR1 could coordinately modulate RNA targets. Successful completion of these aims will further our knowledge of ZC3H14-associated intellectual disability and may also provide invaluable insights into the pathology of the most common genetic cause of intellectual disability, Fragile-X Syndrome, through our analysis of the dNab2/dFMR1 interaction.
This proposal seeks to understand the function of a protein that is altered in patients afflicted with intellectual disability, which was previously referred t as mental retardation. This protein regulates RNA messages that carry genetic information from the nucleus into the cytoplasm of cells and influences how the information in these messages is decoded. Our proposed studies will provide insight into how expression of RNA messages is regulated in cells and also help us learn what is wrong in the brains of patients suffering from this form of intellectual disability.