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

项目摘要 神经元具有高度专业化的结构和功能；它们的遗传信息通常位于一个很大的 距离信息传输地点的距离，他们必须动态改变突触 蛋白质组对神经活动的反应。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 遗传信息被展开和处理是细胞多样性的基础。在这种观点下，仔细审查 RNA新陈代谢的调节，前mRNA基因拷贝如何交替剪接和多腺苷化，编辑， 本地化并受翻译监管，将为理解复杂的流程提供新的途径，如 作为学习和记忆基础的突触可塑性。这一概念驱使分子神经学家寻找 用于定位和调节突触RNA的因子。我们才刚刚开始汇编这些关键因素的清单 调节因子，如RNA结合蛋白(RBPs)，特别是在海马神经元突触中 与记忆有关，到目前为止，人们对这些因素如何调节RNA知之甚少。我们的实验室有 最近建立了一种活体内限制性商业惯例的细胞特异性交联免疫沉淀(CLIP)新平台 小鼠的大脑(CTAG-CLIP)，这进一步加深了我们对细胞特异性调控功能的理解 限制性商业惯例；然而，这项技术仅限于观察稳定状态的RNA调节。在这里，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 被称为opto-CLIP的方法将结合CTAG-CLIP提供的细胞类型特定分辨率与 光遗传学前所未有的精确度，实现了对特定神经元的非侵入性光学控制。一次 建立后，我们将通过在不同的亚细胞隔间执行光剪裁来提高细胞分辨率 目的：评估局部RNA调节与神经功能的关系。这项研究将加深我们对 RBP介导的RNA调节，增强了我们对RNA代谢在突触可塑性中的作用的了解，以及 为神经疾病的病理机制提供了新的见解。总而言之，我是 我相信我研究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调控在突触可塑性中的作用。

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.