Mechanism of mRNA Localization and Localized Translation in Neurons

摘要 神经元是大脑的基本细胞单位。为了让神经元正常工作，它们必须 具有可塑性，并且能够不断地改变以响应刺激、形成和 稳定新的连接。这一过程需要将蛋白质添加到新的 突触接触，而这反过来又是由于将mRNA靶向到这些部位 活动。这是学习和记忆的机制基础，因为突触是 由对刺激作出反应的蛋白质的产生稳定的，这些刺激对 它的结构完整性。这种信使核糖核酸是如何在神经元中被调节以产生正确的蛋白质的 在正确的地点和时间是我们多年来调查的主题 这笔资金。该提案利用了我们在上一个资助期生成的工具 来解决树突中的mRNA是如何被调控的。其中一个工具是鼠标，在那里我们可以 已经用荧光标记标记了β-肌动蛋白基因，以跟随单个mRNA进入 活的神经元。我们已经发现，信使核糖核酸在旅行时是以惰性形式包裹起来的。 在树枝状晶体中。当它靠近受刺激的树突棘突时， 它展开其RNA有效载荷，产生大量蛋白质，但随后又回到休眠状态 16分钟后进入状态。信使核糖核酸位于上次被刺激的地方。 几个小时，等待下一个信号，在那里它将启动另一轮蛋白质。在这 通过这种方式，突触联系建立起来，与学习和记忆范式一致 这依赖于重复的刺激。如果没有进一步的激活信号，则mRNA 继续它的搜索，以短暂的进程运动，被几个周期打断 扩散。目前的提案是对发现的特定蛋白质的后续研究 结合到负责将其引导到其位置的mRNA邮政编码(邮政编码结合蛋白， ZBP1)，将信使核糖核酸锚定在刺激部位。我们构建的模型 表明该信使核糖核酸翻译成进一步的刺激，我们打算将重点放在 通过描述这些事件和蛋白质的动力学来调节 在这些活动中发挥作用。到目前为止，我们已经研究了β-肌动蛋白mRNA，因为 肌动蛋白是细胞和突触中的主要结构蛋白。然而， 导致复杂结构(如突触)的调控机制必须 协调多种蛋白质的表达。出于这个原因，我们构建了 另一只带有对学习重要的信使核糖核酸的小鼠，Arc，已经被标记了一个 不同的荧光标记。在这个提案中，我们将描述β-肌动蛋白的mRNAs 和Arc一起在小鼠中，这两个mRNAs都可以被不同的 彩色荧光染料。我们最终提出了第三个针对CaMKIIα的杂交颜色基因， 突触中的一种必需蛋白质。我们的目标是发现管理 不同的mRNAs对神经元刺激激活反应的调节： 它们合成的时间，定位于树突到激活的脊椎，它们的翻译 抑制、激活和最终降解，以及与之相关的一些蛋白质 这些事件中的每一个。工具现已就位，用于描述这些步骤中的每个步骤 活神经元中的单分子水平。

ABSTRACT The neuron is the basic cellular unit of the brain. For neurons to work properly, they must be plastic and constantly capable of changing in response to stimuli, forming and stabilizing new connections. This process requires proteins to be added to the new synaptic contact, and this in turn results from the targeting of mRNA to these sites of activity. This is the mechanistic basis of learning and memory since the synapse is stabilized by the production of proteins in response to stimulation that are important for its structural integrity. How this mRNA is regulated in neurons to make the right protein at the right place and time has been the subject of our investigations over the years of this funding. This proposal exploits the tools we generated during the last funding period to address how mRNA is regulated in dendrites. One of these tools is a mouse where we have tagged the β-actin gene with a fluorescent marker to follow individual mRNAs in live neurons. We have found that the mRNA is encased in an inert form as it travels around in the dendrite. When it comes into the proximity of a stimulated dendritic spine, it unfurls its RNA payload and makes a burst of protein, but then returns to a dormant state after 16 minutes. The mRNA sits at the place where it was last stimulated for hours, awaiting the next signal, wherein it will initiate another round of proteins. In this way, the synaptic contact is built up, consistent with a learning and memory paradigm that relies on repetitive stimulation. If there are no further activating signals, the mRNA continues its search, moving in short processive movements broken by periods of diffusion. The current proposal follows up on the discovery of the particular protein that binds to the mRNA zipcode responsible for directing it to its site (zipcode binding protein, ZBP1), anchors the mRNA at the site of stimulation. The model we have constructed suggests that the mRNA translates upon a further stimulation and we intend to focus on this point of regulation by describing the kinetics of these events and the proteins that play a role in these events. Up to this point we have investigated β-actin mRNA because actin is the major structural protein in cells, and in the synapse as well. However the regulatory mechanism leading to a complex structure such as a synapse must orchestrate the expression of many proteins. For this reason, we have constructed another mouse with an mRNA important for learning, Arc, that has been tagged with a different fluorescent marker. In this proposal, we will characterize the mRNAs for β-actin and Arc in mice together where both mRNAs are individually detectable by different colored fluorochromes. We propose eventually a third hybrid-color mRNA for CaMKIIα, an essential protein in synapses. Our goal is to uncover the mechanisms that govern the regulation of different mRNAs in response to stimulatory activations of the neuron: the timing of their synthesis, localization into dendrites to activated spines, their translational repression, activation and eventual degradation, and some proteins associated with each of these events. The tools are now in place to characterize each of these steps at the single molecule level in live neurons.