The delivery of mRNA from the nucleus to distal dendritic sites is imperative for localized gene expression. This is required for protein synthesis-dependent changes in synapse plasticity in response to neuro- transmission. Several mRNA-binding proteins (mRBPs) have been implicated in this process, and one is the fragile X mental retardation protein, FMRP, which is absent in fragile X syndrome (FXS). This results in cognitive impairment and autism in humans and altered protein synthesis-dependent plasticity in a mouse model of FXS. Most attention has focused on FMRP in translational regulation at the synapse, but this has only been explored biochemically, while the role of FMRP in mRNA targeting to the synapse has not been addressed. This proposal will test the hypothesis that FMRP acts as a molecular adapter between target mRNAs to which it binds and the molecular motor machinery that drives transport into dendrites. Strong preliminary data demonstrates a role for FMRP in motor-dependent, stimulus-induced mRNA localization in fixed neurons. To more directly determine the role of FMRP in the transport and translation of mRNA in vivo, we have developed methods to visualize the dynamic movements of mRNAs in real time, coupled with the ability to measure translation into protein by fluorescence microscopy.
Specific Aim 1 will quantitatively compare the dynamics of FMRP target mRNAs critical for synaptogenesis and plasticity in dendrites of neurons cultured from wild-type and FMRP-knockout (KO) mice.
Specific Aim 2 will visualize and measure translation of these mRNAs over discrete time periods to determine if they are altered in KO neurons. These assays will be examined in response to activation of group 1 metabotropic glutamate receptors, which increases transport of FMRP and target mRNAs into dendrites, and is known to trigger rapid translation.
Specific Aim 3 will identify domains of FMRP required for mRNA transport and translation through functional rescue in KO neurons. We predict that mRNA transport and translation rates will be altered in the absence of FMRP.
Specific Aim 4 is to develop the PI to obtain R01 funding. FXS is the most common inherited form of mental retardation in humans and currently the leading known cause of autism. The visualization and quantification of mRNA transport and translation in living cells will provide novel insight into the spatial and temporal regulation of gene expression during neuronal development and the role of FMRP in this process.
| Dictenberg, Jason (2012) Genetic encoding of fluorescent RNA ensures a bright future for visualizing nucleic acid dynamics. Trends Biotechnol 30:621-6 |
| Dictenberg, Jason B; Swanger, Sharon A; Antar, Laura N et al. (2008) A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome. Dev Cell 14:926-39 |
