Presynaptic RNA binding protein regulation of learning and memory in C. elegans

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PROJECT ABSTRACT Local protein synthesis in neuronal synapses is necessary for several aspects of learning and memory, including synaptic plasticity and consolidation of long-term memories. Studies of local protein synthesis have long suggested that translation occurs in presynaptic areas, but presynaptic protein synthesis has only been widely accepted by the scientific community in the last decade. As a result, virtually all studies looking at learning and memory have focused on post-synaptic protein synthesis. However, presynaptic protein synthesis also occurs during learning and memory across animal species, raising the question of how presynaptic protein synthesis is regulated and how this contributes to learning and memory. Thus, our lab set out to identify presynaptically localized transcripts and study their role in learning and memory. Subcellular sequencing of neuronal somas and synapses in the nematode worm Caenorhabditis elegans showed that presynaptic areas are enriched for mRNA transcripts encoding RNA binding proteins (RBPs). Studies in mammals have shown that RBPs are upregulated in presynapses following memory training in rats, and post-synaptic RBPs are key regulators of post-synaptic protein synthesis. Thus, RBPs may be key regulators of presynaptic protein synthesis during learning and memory, but further investigation is needed to test if this is the case. To this end, I propose aims to understand how presynaptic RBPs contribute to cognitive function and memory . I will use the nematode worm C. elegans for the proposed experiments because it is the only organism that has a well-defined presynaptic transcriptome, advanced genetic tools, and exhibits evolutionarily conserved memory. In Aim 1, I will test the hypothesis that loss of conserved, presynaptically enriched RBPs will modulate learning and memory. Specifically, I will knock down presynaptically enriched RBPs and test the effect of RNAi knockdown on positive olfactory associative memory in C. elegans. Preliminary data suggests that these tests will reveal presynaptic RBPs as novel memory regulators. In Aim 2, I will mechanistically study how RBPs can modulate learning and memory by identifying the mRNAs bound by a known memory-regulating RBP, PUF-8. I will then test the functional consequences of these mRNA targets on learning and memory. In Aim 2.1, I will perform eCLIP-seq on PUF-8 before and after memory training to identify mRNAs that are bound by PUF-8 in a memory- dependent manner, which are likely downstream memory-regulating genes. I will then validate my eCLIP findings by ensuring that these mRNA targets co-localize with PUF-8 in presynapses (Aim 2.2) and have functional consequences on learning and memory (Aim 2.3). Combined, the proposed studies will provide novel insight into the role of presynaptic transcripts in learning and memory. More specifically, because RBPs are understudied in terms of presynaptic translation, this proposal will address the mechanism of RBP control of downstream mRNAs during learning and memory.