A ribosome interactome that regulates local translation and neural function

调节局部翻译和神经功能的核糖体相互作用组

• 批准号: 10632135
• 负责人: Maria Barna
• 金额: $ 19.68万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10632135
• 关键词: 5' Untranslated Regions; Address; Affect; Axon; Binding; Binding Proteins; Biological; Biotin; Biotinylation; Brain; Cell Volumes; Cells; Cellular biology; Childhood; Complex; Cues; Defect; Dendrites; Development; Disease; Embryonic Development; Endowment; Gene Expression; Genetic study; Grant; Human; Label; Length; Link; Location; Long-Term Potentiation; Loss of Heterozygosity; Maintenance; Mediating; Memory; Mental Depression; Messenger RNA; Morphology; Mus; Mutation; Neurodevelopmental Disorder; Neurons; Neurophysiology - biologic function; Organelles; Peptide Initiation Factors; Physiological; Protein Biosynthesis; Proteins; Proteome; RNA Helicase; Resolution; Ribosomal Interaction; Ribosomal Proteins; Ribosomes; Role; Shapes; Signal Transduction; Specificity; Stimulus; Structure; Subcellular Spaces; Synapses; Synaptic Transmission; Synaptic plasticity; System; Technology; Time; Trans-Activators; Transcript; Translating; Translations; Work; brain health; experimental study; extracellular; helicase; in vivo; insight; mRNA Translation; neural; neurodevelopment; new technology; novel; optogenetics; postsynaptic; presynaptic; response; spatiotemporal; tool; tool development

A central question in cell biology is how gene expression is spatially and temporally regulated in response to stimuli. Neurons are particularly mystifying due to their complex morphology, wherein dendrites and axons that comprise most of the cell volume extend great distances (>10 mm in length) from the cell body. Paradoxically, neurons must respond in a fast and selective manner to accurately transmit synaptic signals across these distances to neighboring cells. In vivo genetic studies have demonstrated a clear requirement for newly synthesized proteins to drive long-term potentiation and depression, synaptic plasticity, and memory formation Indeed, all the components necessary for translation including mRNAs, ribosomes, and initiation factors, are localized within axons and dendrites. This raises the question: how are specific subsets of mRNAs translationally regulated in a selective, fast, and spatially localized manner to propagate distinct signals within neurons? Intriguingly, trans-acting factors known as ribosome-associated proteins (RAPs) have emerged as critical players in regulating translational specificity and subcellular localization that can rapidly fine-tune translation in response to extracellular signals. However, we lack the technologies to be able to precisely isolate and analyze the translational machinery at discrete locations within neurons. In this grant, we will apply new technologies to mark and characterize ribosomes in distinct subdomains of neurons for the first time. We will also directly delineate how RAP binding to the ribosome endows greater specificity in translational control to reflect unique cellular needs and diversity in subcellular space in neurons. In Aim1 we will develop a new technology known as ALIBi (AviTag-specific Location-restricted Inducible Biotinylation), which enables proximity-dependent biotin labeling for the isolation of ribosomes in a spatiotemporally targeted manner. With this technology we will be able to identify RAPs and study localized translation at an unprecedented subcellular resolution in a tunable and highly specific fashion. In Aim2 we will characterize a novel RAP that encodes an ATP-dependent helicase that is present on neuronal ribosomes. Neurons translate some of the longest transcripts in the body containing highly structured 5’UTRs that may require helicase activity for their translation. Here, we will address the outstanding question of whether RAP binding to neural ribosomes endows greater specificity to translational control. Together, this work will uncover the functional consequences of RAP-ribosome interactions with respect to localized translation and neural development utilizing new technologies that for the first time enable us to directly probe neural ribosomes and their functions in localized translational control.

细胞生物学中的一个核心问题是基因表达如何在空间和暂时调节。 刺激。神经元由于其复杂的形态而尤其是神秘的，其中树突和轴突是 包括大部分细胞体积延伸距细胞体的距离（长度> 10 mm）。矛盾的是， 神经元必须快速，选择性地做出响应，以准确地传输合成信号 邻近细胞的距离。体内遗传研究表明对新近的需求明确 合成的蛋白质以驱动长期增强和抑郁，突触可塑性和记忆形成 实际上，翻译所需的所有组成部分，包括mRNA，核糖体和主动因素，都是 位于轴突和树突中。这提出了一个问题：mRNA的特定子集如何 以选择性，快速和空间本地化的方式进行翻译调节，以传播不同的信号 神经元？有趣的是，被称为核糖体相关蛋白（RAP）的反式作用因子已出现为 控制翻译特异性和亚细胞定位的关键参与者可以快速微调 响应细胞外信号的翻译。但是，我们缺乏能够精确的技术 隔离和分析神经元内离散位置的翻译机制。在这笔赠款中，我们将申请 首次在神经元的不同子域中标记和表征核糖体的新技术。我们 还将直接描述说唱如何与核糖体结合在翻译控制中更特异性 反映神经元中亚细胞空间中独特的细胞需求和多样性。在AIM1中，我们将开发一个新的 被称为Alibi的技术（特定于AVITAG的位置限制诱导生物素化），可实现 接近依赖性生物素标记，用于在空间靶向中分离核糖体 方式。通过这项技术，我们将能够识别说唱并研究 以可调且高度特定的方式以前所未有的亚细胞分辨率。在AIM2中，我们将表征一个 编码神经元核糖体上存在的ATP依赖性解旋酶的新型RAP。神经元翻译 人体中一些最长的成绩单，其中包含高度结构化5'UTR，可能需要解旋酶 他们翻译的活动。在这里，我们将解决说唱是否与中立结合的杰出问题 核糖体赋予翻译控制更特异性。这项工作将共同揭示功能 说唱 - 核糖体相互作用在局部翻译和神经发育方面的后果 使用新技术首次使我们能够直接探测神经核糖体及其功能 在局部翻译控制中。