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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

细胞生物学的一个中心问题是基因表达如何在空间和时间上调节以响应神经元由于其复杂的形态而特别神秘，但是树突和轴突矛盾的是，大部分细胞体积从细胞体延伸很远的距离（长度> 10毫米）。神经元必须以快速且有选择性的方式做出反应，以准确地在这些神经元之间传递突触信号。体内遗传学研究已经证明了对新细胞的明确要求。合成蛋白质以驱动长时程增强和抑制、突触可塑性和记忆形成事实上，翻译所需的所有成分，包括 mRNA、核糖体和起始因子，都是这提出了一个问题：mRNA 的特定子集是如何定位的。以选择性、快速和空间局部化的方式进行翻译调节，以在内部传播不同的信号有趣的是，被称为核糖体相关蛋白（RAP）的反式作用因子已经出现调节翻译特异性和亚细胞定位的关键参与者，可以快速微调然而，我们缺乏能够精确翻译的技术。在这笔资助中，我们将应用分离和分析神经元内离散位置的翻译机制。首次使用新技术来标记和表征神经元不同子域中的核糖体。还将直接描述 RAP 与核糖体的结合如何赋予翻译控制更大的特异性为了反映神经元亚细胞空间的独特细胞需求和多样性，我们将在 Aim1 中开发一种新的。称为 ALIBi（AviTag 特定位置限制诱导生物素化）的技术，该技术使邻近依赖性生物素标记用于时空靶向分离核糖体借助这项技术，我们将能够快速识别 RAP 并研究本地化翻译。在 Aim2 中，我们将以可调节且高度特异性的方式获得前所未有的亚细胞分辨率。编码存在于神经元核糖体上的 ATP 依赖性解旋酶的新型 RAP 神经元翻译。体内一些最长的转录本包含可能需要解旋酶的高度结构化的 5’UTR 在这里，我们将解决 RAP 是否与神经结合这一悬而未决的问题。核糖体赋予翻译控制更大的特异性，这项工作将揭示其功能。 RAP-核糖体相互作用对局部翻译和神经发育的影响利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本地化翻译控制中。