Functional Analysis of Neuronal RNA Transport Particles

mRNA localization and regulated translation are well-established mechanisms for the post-transcriptional control of gene expression. These processes act in concert to promote spatially and temporally precise translation of proteins at subcellular sites. Localized translation is crucial in developmental processes, and also for the function of mature polarized cells including neurons, glia, fibroblasts, and epithelial cells. Defects in RNA localization and other post-transcriptional regulatory processes can have severe consequences for cellular function. These defects underlie several human diseases including fragile X mental retardation, spinal muscular atrophy, paraneoplastic opsoclonus-myoclonus, and myotonic dystrophy. Functional studies of RNA localization in various organisms have revealed some generally conserved features of the process including a requirement for cis-acting localization signals in localized transcripts, recognition of these signals by RNA-binding proteins, packaging into ribonucleoprotein particles, transport along the cytoskeleton, and the involvement of motor proteins. However, the actual molecular machinery involved is largely unknown. Our goal is to identify and study the molecular components that link the recognition of localized neuronal RNAs to directed movement along the cytoskeleton, and that coordinate the translation of localized messages with their translocation. We will expand upon previous work in our laboratory utilizing biochemical fractionation techniques to isolate RNA transport particles from rat brain. We will use the known particle components Staufen1, Staufen2, and Barentsz as markers and molecular `handles` for purification. We will identify protein and RNA components of these particles, testing for known candidate molecules and identifying novel factors. We will proceed to confirm the relevance of these components for RNA localization using both in vivo and in vitro assays. We are concentrating on the isolation of endogenous localization complexes from brains in order to specifically understand post-transcriptional control mechanisms underlying neuronal functions, e.g. dendritic spine morphogenesis. We are initially focused on elucidating basic, core features of the localization process that are likely to be conserved between cell types and species.