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是如何在空间和时间上调节的，以响应 刺激物。神经元由于其复杂的形态而特别令人困惑，其中树突和轴突 构成细胞体积的大部分延伸到细胞体很远的距离(10毫米长)。矛盾的是， 神经元必须以快速和选择性的方式做出反应，才能准确地通过这些区域传递突触信号。 到相邻单元格的距离。体内遗传学研究表明，新的 合成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，翻译所需的所有成分，包括mRNA、核糖体和启动因子，都是 定位于轴突和树突内。这就提出了一个问题：mRNAs的特定子集是如何 以选择性、快速和空间局部化的方式进行翻译调节，以在 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为 在调节翻译特异性和亚细胞定位方面发挥关键作用，可以快速微调 对细胞外信号作出反应的翻译。然而，我们缺乏能够准确地 分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亚域中标记和表征核糖体的新技术。我们 还将直接描述RAP与核糖体结合如何在翻译控制中赋予更大的特异性 以反映神经元中独特的细胞需求和亚细胞空间的多样性。在Aim1中，我们将开发一种新的 被称为AliBI(AviTag特定位置限制的可诱导生物素化)的技术，它能够 邻近生物素标记用于时空靶向核糖体的分离 举止。有了这项技术，我们将能够识别说唱并研究本地化翻译 史无前例的亚细胞分辨率，可调且高度具体。在AIM2中，我们将描述一个 编码神经元核糖体上存在的一种依赖于ATP的解旋酶的新型RAP。神经元翻译 体内一些最长的转录本，含有可能需要解旋酶的高度结构化的5‘UTRs 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是否与神经结合 核糖体赋予翻译控制更大的特异性。总而言之，这项工作将揭示 RAP-核糖体相互作用对局部翻译和神经发育的影响 利用首次使我们能够直接探测神经核糖体及其功能的新技术 在局部的平移控制中。

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.