Combinatorial use of translational co-factors for cell type-specific regulation during neuronal morphogenesis in Drosophila

AbstractThe translational regulators Nanos (Nos) and Pumilio (Pum) work together to regulate the morphogenesis of dendritic arborization (da) neurons of the Drosophila larval peripheral nervous system. In contrast, Nos and Pum function in opposition to one another in the neuromuscular junction to regulate the morphogenesis and the electrophysiological properties of synaptic boutons. Neither the cellular functions of Nos and Pum nor their regulatory targets in neuronal morphogenesis are known. Here we show that Nos and Pum are required to maintain the dendritic complexity of da neurons during larval growth by promoting the outgrowth of new dendritic branches and the stabilization of existing dendritic branches, in part by regulating the expression of cut and head involution defective. Through an RNA interference screen we uncover a role for the translational co-factor Brain Tumor (Brat) in dendrite morphogenesis of da neurons and demonstrate that Nos, Pum, and Brat interact genetically to regulate dendrite morphogenesis. In the neuromuscular junction, Brat function is most likely specific for Pum in the presynaptic regulation of bouton morphogenesis. Our results reveal how the combinatorial use of co-regulators like Nos, Pum and Brat can diversify their roles in post-transcriptional regulation of gene expression for neuronal morphogenesis

Argonaute2 Suppresses Drosophila Fragile X Expression Preventing Neurogenesis and Oogenesis Defects

Fragile X Syndrome is caused by the silencing of the Fragile X Mental Retardation gene (FMR1). Regulating dosage of FMR1 levels is critical for proper development and function of the nervous system and germ line, but the pathways responsible for maintaining normal expression levels are less clearly defined. Loss of Drosophila Fragile X protein (dFMR1) causes several behavioral and developmental defects in the fly, many of which are analogous to those seen in Fragile X patients. Over-expression of dFMR1 also causes specific neuronal and behavioral abnormalities. We have found that Argonaute2 (Ago2), the core component of the small interfering RNA (siRNA) pathway, regulates dfmr1 expression. Previously, the relationship between dFMR1 and Ago2 was defined by their physical interaction and co-regulation of downstream targets. We have found that Ago2 and dFMR1 are also connected through a regulatory relationship. Ago2 mediated repression of dFMR1 prevents axon growth and branching defects of the Drosophila neuromuscular junction (NMJ). Consequently, the neurogenesis defects in larvae mutant for both dfmr1 and Ago2 mirror those in dfmr1 null mutants. The Ago2 null phenotype at the NMJ is rescued in animals carrying an Ago2 genomic rescue construct. However, animals carrying a mutant Ago2 allele that produces Ago2 with significantly reduced endoribonuclease catalytic activity are normal with respect to the NMJ phenotypes examined. dFMR1 regulation by Ago2 is also observed in the germ line causing a multiple oocyte in a single egg chamber mutant phenotype. We have identified Ago2 as a regulator of dfmr1 expression and have clarified an important developmental role for Ago2 in the nervous system and germ line that requires dfmr1 function

Modulation of ADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein

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