Post-synaptic neuronal responsiveness results from the modulation of neuronal dendritic function. It has become clear over the last several years that various mRNAs are targeted to dendrites where they can be locally translated in response to presynaptic stimulation. The targeting of RNAs into dendrites occurs through the direct association of RNA binding proteins (RBP) with the RNA being transported. Generally, RBPs not only function as mediators of subcellular localization but also act as chaperones to control the metabolism of RNAs, including transcription of genes, the maturation of RNAs, the nucleo-cytoplasmic transport of RNAs, the translation of RNAs and the degradation of RNAs. The study of populations of RNAs and their regulatory RBPs has been called Ribonomics. In this proposal we propose to assess the Ribonomics of dendritic functioning. To do this we will assess three Specific Aims that directly derived from our preliminary data. In particular we will 1) identify the RBPs that bind to three RNAs that are targeted to and differentially translated in neuronal dendrites, those that encode the GluR2, GluR4 and the NMDA-R1 receptor subunit proteins. The identification of the RBPs will be performed using a novel technology that we have called the PAIR technology. After the RBPs are identified, antibodies directed against these RBPs will be used in the antibody positioned RNA amplification (APRA) methodology to characterize the family of other RNAs that bind to the specific RBPs. The combination of the PAIR and APRA methodologies will permit us to assess changes in these RNAs, their associated RBPs and the RBP co-regulated RNAs during the aging process. Secondly, we have recently shown that dendrites have the capacity to modify RNAs though extra-nuclear RNA splicing. We will continue to investigate this phenomena by identifying endogenous RNAs that can be locally spliced in dendrites. And finally, using a variety of biochemical and molecular approaches (including a novel subcellular transfection methodology) a dendrically translated RNA that gives rise to neuronal cell death. We will assess both fundamental questions of cell death related to this molecule as well as determine whether there is a potential role for this cell death paradigm in Parkinson's Disease and physiological aging. At the conclusion of these studies important roles of RBPs and their cargoes in modulating dendritic function will have been elucidated.
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