The long term objective of the project is to examine the mechanisms underlying mRNA sorting and transport in neurons. In dendrites, these mRNAs are thought to play a key role in providing a substrate for local synthesis of key protein constituents of synapses involved in remodeling the neuron morphology after activation of the synapse. The mRNA for the immediate early gene Arc will be used for these studies. The assessment of IEG mRNAs allows an evaluation of the differential sorting of newly synthesized mRNAs and an analysis of the directionality and kinetics of movement of the mRNAs from their site of synthesis. The examination of the kinetics of movement is not possible with any constitutively expressed mRNA, including mRNA constructs introduced into neurons in vitro by using transfection techniques. At present there is little information about the key players involved in RNA localization to dendrites, but transport in the hippocampus of rats has been extensively studied. Cis acting RNA Transport Sequences (RTS) will be identified using transfectable chimeric constructs with various regions of the Arc gene fused to a reporter gene. In situ hybridization assays of transfected neurons will be performed using antisense RNA probes to identify the location of the reporter transcripts. Various deletions will be made in the Arc regions until the RTS is identified. Extracts made from rat hippocampii will be used to identify and purify the proteins that bind to these sequence elements. These findings will extend our understanding of the mechanisms that lead to a differential localization of mRNAS and provide new clues about the nature of the transport mechanisms that deliver mRNAs into dendrites. Although it remains to be determined if the trafficking mechanisms that operate for IEG mRNAs are the same or different from the mechanisms that operate for other differentially sorted mRNAs, analysis of the sorting of IEGs provides a currently unique level of access to the dynamic process of mRNA localization.
