To begin to understand the mechanisms underlying dendritic mRNA targeting, we have cloned a gene from a rat hippocampal cDNA library that is related to Drosophila staufen, a gene critical for the asymmetric targeting of specific mRNAs in oocytes and neuroblasts. In situ hybridization experiments indicate that rat staufen is expressed in the hippocampus, including the CA1-CA3 and dentate gyrus regions. Preliminary results of in vitro RNA binding assays suggest that the recombinant rat Staufen protein can bind to multiple mRNAs present in the dendrites of hippocampal neurons, including CamKII, TrkB and Arc. Transfection of hippocampal neurons with a GFP-Staufen fusion construct resulted in a punctate GFP pattern in dendrites, suggesting a dendritic distribution for rat Staufen.
Immunohistochemistry suggests a similar distribution.
These results are consistent with the hypothesis that rat Staufen is associated with dendritic RNAs in vivo and is may be involved in their targeting to dendritic compartments. We are also interested in visualizing, directly, the synthesis of proteins in dendrites. To this end, we have constructed a cDNA construct encoding a destabilized variant of EGFP flanked by the 5' and 3' UTR regulatory regions of the CamKII- gene. The cDNA constructs were transfected into cultured hippocampal neurons using Biolistics. We are currently examining the effects of synaptic activity and plasticity on translation of the localized GFP message.