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

项目摘要 神经元突触中的局部蛋白质合成是学习和记忆的几个方面所必需的， 包括突触的可塑性和长期记忆的巩固。对局部蛋白质合成的研究有 Long认为翻译发生在突触前区域，但突触前蛋白质的合成 在过去的十年里被科学界广泛接受。结果，几乎所有的研究都着眼于学习 和记忆都集中在突触后蛋白质的合成上。然而，突触前蛋白质的合成也 在动物物种的学习和记忆过程中发生，提出了突触前蛋白是如何 合成是受调控的，以及它是如何促进学习和记忆的。因此，我们的实验室着手确定 突触前定位转录本，研究它们在学习和记忆中的作用。亚细胞测序 线虫线虫的神经元胞体和突触显示突触前区域 富含编码RNA结合蛋白(RBPs)的mRNA转录本。对哺乳动物的研究表明 大鼠记忆训练后，突触前RBPs上调，突触后RBPs是关键 突触后蛋白质合成的调节因子。因此，RBPs可能是突触前蛋白合成的关键调节因子。 在学习和记忆过程中，但如果是这样，还需要进一步的调查。为此，我建议 旨在了解突触前RBPs如何有助于认知功能和记忆 。我会用线虫 蠕虫线虫用于拟议的实验，因为它是唯一具有明确定义的 突触前转录组，先进的遗传工具，并表现出进化保守的记忆。在目标1中，我 将检验这样一种假设，即失去保守的、突触前丰富的RBPs将调节学习和 记忆。具体地说，我将击倒突触前浓缩的限制性商业惯例，并测试RNAi敲除的效果 线虫正嗅觉联想记忆的研究。初步数据显示，这些测试将揭示 突触前限制性商业惯例作为新的记忆调节剂。在目标2中，我将机械地研究限制性商业惯例如何调节 通过识别已知的记忆调节RBP PUF-8结合的mRNAs来学习和记忆。那我会的 测试这些信使核糖核酸靶标对学习和记忆的功能影响。在Aim 2.1中，我将表演 记忆训练前后PUF-8上的eCLIP-seq以识别内存中由PUF-8结合的mRNAs 依赖的方式，这可能是下游的记忆调节基因。然后我将验证我的eCLIP调查结果 通过确保这些mRNA靶点与PUF-8在突触前(目标2.2)共定位并具有功能 对学习和记忆的影响(目标2.3)。总而言之，拟议的研究将提供对 突触前转录产物在学习和记忆中的作用。更具体地说，因为限制性商业惯例在 在突触前翻译方面，本提案将解决RBP对下游mRNAs的控制机制 在学习和记忆过程中。

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.