Abstract
Fragile X mental retardation protein (FMRP) plays a vital role in mRNA trafficking and translation inhibition to regulate the synthesis of local proteins in neuronal axons and dendritic terminals. However, there are no reports on microRNA (miRNA)-mediated regulation of FMRP levels in Drosophila. Here, we aimed to identify miRNAs regulating FMRP levels in Drosophila. Using online software, we predicted and selected 11 miRNAs potentially acting on the Drosophila fragile X mental retardation 1 (dFMR1) transcript. These candidates were screened for modulation of dFMR1 transcript levels at the cellular level using a dual luciferase reporter system. In addition, we constructed a transgenic Drosophila model overexpressing miR-219 in the nervous system and quantified dFMRP by western blotting. The neuromuscular junction phenotype in the model was studied by immunofluorescence staining. Among the 11 miRNAs screened, miR-219 and miR-960 reduced luciferase gene activity by binding to the 3'-UTR of the dFMR1 transcript. Mutation of the miR-219 or miR-960 binding sites on the transcript resulted in complete or partial elimination of the miRNA-induced repression. Western blots revealed that dFMRP expression was decreased in the miR-219 overexpression model (Elav>miR-219). Drosophila larvae overexpressing miR-219 showed morphological abnormalities at the neuromuscular junction (increased synaptic boutons and synaptic branches). This finding is consistent with some phenotypes observed in dfmr1 mutants. Our results suggest that miR-219 regulates dFMR1 expression in Drosophila and is involved in fragile X syndrome pathogenesis. Collectively, these findings expand the current understanding of miRNA-mediated regulation of target molecule-related functions.
