A FUNCTIONAL ANALYSIS OF RNA-BINDING PROTEINS IN POST-TRANSCRIPTIONAL REGULATION DURING NEURONAL DENDRITE MORPHOGENESIS

Abstract

Post-transcriptional regulation enables precise spatiotemporal control of gene expression, which is critical for generating morphological and functional asymmetries in many developmental processes. Such regulation is particularly important in highly polarized cells like neurons by facilitating on-site, on-demand protein synthesis. RNA-binding proteins (RBPs) play versatile roles in multiple aspects of RNA metabolism. Their control of RNA behavior and function make them key regulators in dendrite development. Our studies focused on Fragile X mental retardation protein (FMRP), a highly conserved RBP that has important functions in the nervous system and loss of which causes Fragile X syndrome (FXS). Using Drosophila sensory neurons as a model system, we elucidated the regulatory roles of FMRP in neuronal dendrite morphogenesis and showed that it regulates dendritic growth in part through dendritic transport and translation control of profilin/chickadee mRNA. We confirmed the requirement of the FMRP KH1 and KH2 domains, but not the RGG box, in FMRP-mediated dendritic regulation. Notably, the KH2 domain is also indispensable for FMRP granule formation and profilin/chickadee mRNA association, connecting FMRP granule formation to its dendritic function. FXS has been connected to microRNA pathway dysfunction in axonal elongation and synaptic plasticity and we demonstrated the involvement of a FMRP-associated microRNA, miR-276, in control of dendritic space filling morphology. This occurs through the FMRP-miR-276 regulatory axis rather than the alteration of miR-276 levels. We also identified dCBP/nejire mRNA as a downstream target of this regulatory axis. Our results support a model where FMRP also controls dendritic patterns through miR-276-mediated nej regulation. A separate aspect of this work relates to RNA localization in Drosophila oocytes, with a particular focus on oskar mRNA whose posterior localization is important for abdominal patterning and germ cell formation. Specifically, we characterized the requirements for oskar accumulation in large structures called founder granules during late oogenesis, and determined the involvement of cis-acting elements – the spliced oskar localizaiton element (SOLE) – as well as trans-acting regulatory RBPs, including Hrp48 and Staufen, in this process. With super-resolution microscopy, we observed that founder granules comprise multiple physically distinct oskar transport particles, providing novel insight in oskar posterior enrichment by an entrapment mechanism.