Fragile X syndrome (FXS) is the most common heritable form of intellectual disability and a known genetic cause of autism. This disease arises from a trinucleotide repeat expansion in the 5’ untranslated region (UTR) of the fragile X mental retardation (FMR1) gene, which results in hypermethylation and transcriptional silencing of FMR1 gene expression. Although previous studies suggest that the protein encoded from this locus (FMRP) functions as a translational regulator, a molecular function for FMRP remains to be elucidated. For my thesis, I sought to gain a better understanding of how FMRP regulates gene expression and how absence of FMRP expression gives rise to the phenotypes associated with FXS. To explore how FMRP functions at a molecular level, our lab utilized tandem affinity purification (TAP) followed by mass spectrometry analysis to identify novel proteins that interact with the Drosophila FMRP homolog (dFMR1). Through this screen, our lab pulled out the A-to-I RNA editing enzyme, dADAR, as a protein that interacts with dFMR1. Using several biochemical techniques, we verified this interaction using Drosophila S2 cell culture and in vivo using adult fly head lysates. Because previous studies demonstrated that loss- and gain-of-dFMR1 expression results in morphological defects at the neuromuscular junction (NMJ), we used this system to test for a genetic interaction between dfmr1 and dAdar. We found that like dfmr1 , dAdar activity is required for aspects of NMJ synaptic architecture. We further demonstrated that although dADAR and dFMR1 are both required for normal NMJ morphology, dAdar and dfmr1 single mutants exhibit distinct morphological defects at the NMJ. Genetic epistasis experiments performed with dfmr1 and dAdar mutant alleles suggest that dAdar acts downstream of dfmr1 and that dFMR1 modulates dADAR activity. Because we found that dFMR1 does not regulate dADAR expression, we hypothesized that dADAR function was affected by changes in dFMR1 levels. In support of this, we found that loss- or overexpression of dFMR1 affects editing efficiency on certain dADAR substrates with defined roles in synaptic transmission. These results demonstrate that dFMR1 associates with another post-transcriptional gene regulatory pathway and indicate that proper synaptic architecture of the NMJ requires modulation of dADAR activity by dFMR1
