A ribosome interactome that regulates local translation and neural function

细胞生物学中的一个中心问题是基因表达如何在空间和时间上响应于 刺激。神经元由于其复杂的形态而特别神秘，其中树突和轴突 包括大部分电池体积的电池从电池体延伸很长的距离（长度>10 mm）。巧合的是， 神经元必须以快速和选择性的方式做出反应，以准确地将突触信号传递到这些神经元。 与相邻小区的距离体内遗传学研究表明，新的 合成的蛋白质驱动长时程增强和抑制、突触可塑性和记忆形成 事实上，翻译所必需的所有成分，包括mRNA、核糖体和起始因子，都是 位于轴突和树突内。这就提出了一个问题：特定的mRNA亚类 以选择性、快速和空间局部化的方式进行调节，以在细胞内传播不同的信号。 神经元？有趣的是，被称为核糖体相关蛋白（RAP）的反式作用因子已经出现， 在调节翻译特异性和亚细胞定位，可以快速微调的关键球员 翻译响应细胞外信号。然而，我们缺乏技术， 分离和分析神经元内离散位置的翻译机器。在这份补助金中，我们将申请 新技术首次在神经元的不同子域中标记和表征核糖体。我们 还将直接描述RAP如何与核糖体结合，赋予翻译控制更大的特异性 以反映独特的细胞需求和神经元亚细胞空间的多样性。在AIM 1中，我们将开发一种新的 被称为ALIBi（AviTag特异性位置限制性诱导生物素化）的技术， 邻近依赖性生物素标记用于时空靶向的核糖体分离 方式有了这项技术，我们将能够识别RAP，并在一个特定的环境中研究本地化翻译。 前所未有的亚细胞分辨率在可调和高度具体的方式。在目标2中，我们将描述一个 一种新的RAP，其编码存在于神经元核糖体上的ATP依赖性解旋酶。神经元翻译 体内一些最长的转录本含有高度结构化的5 'UTR，可能需要解旋酶 为他们的翻译活动。在这里，我们将解决一个悬而未决的问题，即RAP是否与神经细胞结合， 核糖体赋予翻译控制更大的特异性。总之，这项工作将揭示功能 RAP-核糖体相互作用对定位翻译和神经发育的影响 利用新技术，我们首次能够直接探测神经核糖体及其功能， 在本地化的翻译控制。

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.