Shedding light on the role of RNA binding protein-mediated RNA regulation in synaptic plasticity

揭示 RNA 结合蛋白介导的 RNA 调节在突触可塑性中的作用

• 批准号: 10285142
• 负责人: Ruth A Singer
• 金额: $ 6.64万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2022-12-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10285142
• 关键词: Affinity Chromatography; Alleles; Alternative Splicing; Axon; Bioinformatics; Biological Assay; Biology; Brain; Breeding; Caliber; Cell Nucleus; Cells; Complex; DNA; Data; Data Set; Dendrites; Disease; Environment; FMR1; Fluorescent in Situ Hybridization; Foundations; Gene Expression; Genes; Genetic Transcription; High-Throughput Nucleotide Sequencing; Hippocampus (Brain); Immediate-Early Genes; Immunoprecipitation; Knowledge; Laboratories; Learning; Light; Lighting; Measures; Mediating; Memory; Messenger RNA; Methodology; Modeling; Molecular; Mus; Neurologic; Neurons; Neuropil; Neurosciences; Optics; Pathologic; Poly A; Population; Process; Protein Biosynthesis; Proteome; RNA; RNA Binding; RNA metabolism; RNA-Binding Proteins; Regulation; Research; Resolution; Rest; Role; Site; Structure; Synapses; Synaptic plasticity; Technology; Testing; Time; Transcript; Translations; Universities; Up-Regulation; calmodulin-dependent protein kinase II; cell type; crosslink; excitatory neuron; experience; experimental study; genetic information; in vivo; insight; interest; mRNA Precursor; mouse model; nervous system disorder; neuronal cell body; novel; novel strategies; optogenetics; protein distribution; relating to nervous system; response; unpublished works

Project Summary Neurons have highly specialized structures and functions; their genetic information is often located a great distance from the sites where information gets transmitted, and they must dynamically alter the synaptic proteome in response to neural activity. These challenges indicate that neurons have evolved unique regulatory mechanisms to meet functional demands. The uniformity of DNA across different cell types suggests that how genetic information is unfolded and processed underlies cellular diversity. In this view, careful examination of the regulation of RNA metabolism, how pre-mRNA gene copies are alternatively spliced and polyadenylated, edited, localized, and translationally regulated, will offer a new avenue towards understanding complex processes, such as synaptic plasticity underlying learning and memory. This notion has driven molecular neuroscientists to search for factors that localize and regulate synaptic RNAs. We are only beginning to compile a list of these key regulators, such as RNA binding proteins (RBPs), particularly in the synapses of hippocampal neurons that are involved in memory, and to date very little is known about how these factors regulate RNA. Our laboratory has recently generated a new platform for cell-specific Crosslinked Immunoprecipitation (CLIP) of RBPs in the living brain of mice (cTag-CLIP), which has furthered our understanding of the cell-specific regulatory functions of RBPs; however, this technology is limited to looking at steady state RNA regulation. Here we described a novel approach to uncover the role of neuronal RBP-mediated RNA regulation in the context of synaptic plasticity. This methodology, termed opto-CLIP, will combine the cell type-specific resolution afforded by cTag-CLIP with the unprecedented precision of optogenetics to achieve non-invasive optical control of specific neurons. Once established, we will increase the cellular resolution by performing opto-CLIP in distinct subcellular compartments to assess local RNA regulation associated with neuronal function. This study will further our understanding of RBP-mediated RNA regulation, enhance our knowledge of the role of RNA metabolism in synaptic plasticity, and provide new insight into the pathological mechanisms underlying neurological disorders. In conclusion, I am confident that my experience studying RNA biology, the Darnell lab's foundation in neuroscience and CLIP, and the rich research environment at Rockefeller University will help me succeed in using optogenetics to study the role of RBP-mediated RNA regulation in synaptic plasticity.

项目概要 神经元具有高度专业化的结构和功能；他们的遗传信息往往位于一个伟大的 距信息传输站点的距离，并且它们必须动态地改变突触 对神经活动做出反应的蛋白质组。这些挑战表明神经元已经进化出独特的调节机制 满足功能需求的机制。不同细胞类型的 DNA 的一致性表明 遗传信息的展开和处理是细胞多样性的基础。鉴于此，仔细审查 RNA 代谢的调节，前 mRNA 基因拷贝如何选择性剪接和聚腺苷酸化、编辑、 本地化和翻译监管将为理解复杂过程提供新途径，例如 作为学习和记忆基础的突触可塑性。这个概念促使分子神经科学家去寻找 用于定位和调节突触 RNA 的因子。我们才刚刚开始编制这些关键的列表 调节因子，例如 RNA 结合蛋白 (RBP)，特别是在海马神经元的突触中 参与记忆，但迄今为止，人们对这些因素如何调节 RNA 知之甚少。我们实验室有 最近开发了一个新平台，用于活体中 RBP 的细胞特异性交联免疫沉淀 (CLIP) 小鼠大脑（cTag-CLIP），进一步加深了我们对细胞特异性调节功能的理解 RBP；然而，这项技术仅限于研究稳态 RNA 调节。这里我们描述了一部小说 揭示神经元 RBP 介导的 RNA 调节在突触可塑性中的作用的方法。这 称为 opto-CLIP 的方法将 cTag-CLIP 提供的细胞类型特异性分辨率与 前所未有的光遗传学精度，实现对特定神经元的非侵入性光学控制。一次 确定后，我们将通过在不同的亚细胞区室中执行 opto-CLIP 来提高细胞分辨率 评估与神经元功能相关的局部 RNA 调节。这项研究将加深我们对 RBP 介导的 RNA 调节，增强我们对 RNA 代谢在突触可塑性中的作用的了解，以及 为神经系统疾病的病理机制提供新的见解。总而言之，我是 对我研究 RNA 生物学的经验、达内尔实验室在神经科学和 CLIP 方面的基础以及 洛克菲勒大学丰富的研究环境将帮助我成功地利用光遗传学来研究 RBP 介导的 RNA 调节在突触可塑性中的作用。