Saturday, May 4, 2024
5/4/2024
PROJECT SUMMARY In this R21 exploratory/developmental grant application, we propose to develop and validate methodologies for in vivo analysis of RNA targeting in specific circuits, as well as investigating the regulation of these RNAs during long-term memory storage (LTM). The significance of RNAs localized to dendrites for local translation is well known. However, current methodologies do not provide accurate identification of RNAs targeted to distinct subcellular compartments. For example, several studies utilizing molecular analysis of microdissected dendrites or neuronal processes have identified RNAs in the distally localized neurons of diverse organisms, including sea slugs and mice. A major caveat of this approach is that the microdissected regions contain dendrites/neuronal processes from several different neurons as well as segments of interneurons and localized non-neuronal cells. Thus, this approach yields RNAs from dendrites of multiple neurons and non- neuronal cells, preventing our ability to identify the neuron-specific, distally targeted RNAs necessary to generate a more profound understanding of RNA localization and LTM. Since, learning and memory involves changes that are circuit-specific, the lack of robust and reliable methodologies to identify circuit-specific, targeted RNAs acts as a critical barrier to our understanding of RNA targeting in vivo, as well as the role that these RNAs play in both in LTM and disorders of memory. To address this critical knowledge gap, we here propose a strategy that starts with the expression of a tagged kinesin that mediates the transport of RNAs in specific neurons followed by immunoprecipitation of kinesin complexes from distinct neuronal projections and identification and rigorous characterization of associated RNAs. We anticipate that our kinesin-based strategy will yield deep insights into in vivo RNA targeting and their modulation by LTM.
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