No abstract

Loss of FMR1 gene function results in fragile X syndrome (FXS), the most common heritable form of intellectual disability. The protein encoded from this locus (FMRP) is an RNA binding protein thought to primarily act as a translational regulator, however recent studies implicate FMRP in other mechanisms of gene regulation. Here, we demonstrate that the Drosophila fragile X homolog (dFMR1) biochemically interacts with the A-to-I RNA editing enzyme, dADAR. We found that dAdar and dfmr1 mutant larvae exhibit distinct morphological neuromuscular junction (NMJ) defects. Epistasis experiments based on these phenotypic differences suggest that dAdar acts downstream of dfmr1 and that dFMR1 modulates dADAR activity. Furthermore, sequence analyses revealed that loss or overexpression of dFMR1 affects editing efficiency on certain dADAR targets with defined roles in synaptic transmission. These results link dFMR1 with the RNA editing pathway and suggest that proper NMJ synaptic architecture requires modulation of dADAR activity by dFMR1.

The 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.

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.

PRIMERFragile X syndrome (FXS) is a cognitive disorder caused by silencing of the fragile X mental retardation 1 gene (FMR1). Since the discovery of the gene almost two decades ago, most scientific contributions have focused on identifying the molecular function of the fragile X mental retardation protein (FMRP) and understanding how absence of FMR1 gene expression gives rise to the disease phenotypes. The use of model organisms has allowed rapid progression in the FXS field and has given insight into the molecular basis of the disease. The mouse and fly FXS models have enabled studies to identify potential targets and pathways for pharmacological treatment. Here, we briefly review the two primary FXS model systems and describe how studies in these organisms have led us closer to therapeutic treatments for patients afflicted with FXS.Disease Models & Mechanisms DMM et al., 2005;Hou et al., 2006). However, other studies suggest that FMRP promotes translation of target mRNAs, such as TrailerHitch and Sod1 transcripts (Monzo et al., 2006;Bechara et al., 2009). In addition, recent studies suggest that FMRP might also regulate transcript stability, such as that of microRNA-124a (miRNA-124a) and PSD-95 (Zalfa et al., 2007;Xu et al., 2008). Thus, the translation and expression of FMRP targets can be either positively or negatively affected by FMRP expression, which indicates that the potential role of FMRP as a translational regulator is much more complex than was originally believed.To complicate further the theory that FMRP acts at the level of translation, biochemical purification of FMRP-bound complexes suggests that FMRP interacts with multiple pathways that also regulate gene expression. For example, FMRP has been found to interact with components of the RNA interference (RNAi) pathway, a gene-silencing mechanism triggered by the presence of double-stranded RNA (dsRNA). Several research groups have shown that FMRP can be purified in complexes with proteins involved in small interfering RNA (siRNA) and miRNA biogenesis (Caudy et al., 2002;Ishizuka et al., 2002;Caudy et al., 2003;Jin et al., 2004). Despite being associated with siRNA pathway members, FMRP has subtle effects on RNAi pathway efficiency and is not required for siRNA biogenesis (Caudy et al., 2002;Ishizuka et al., 2002). Genetic evidence from Drosophila suggests that components of the siRNA pathway and FMRP converge to regulate larval crawling behavior and synaptic growth during development, supporting the theory that FMRP and the RNAi pathway interact (Xu et al., 2004;Pepper et al., 2009). The interaction between FMRP and miRNA pathway components has also revealed interesting biochemical and genetic interactions. FMRP associates with miRNAs and components of the miRNA pathway (Caudy et al., 2002;Ishizuka et al., 2002;Jin et al., 2004;Xu et al., 2008;Edbauer et al., 2010). The FMRPassociated miRNAs have been shown to regulate dendritic branching, which is rescued by genetically reducing FMRP expression and function (Xu et al., 2008;Edbauer et al., 20...