Mechanism of mRNA Localization and Localized Translation in Neurons

摘要 神经元是大脑的基本细胞单位。神经元要正常工作， 是可塑的，能够不断地对刺激做出反应，形成和 稳定新的连接。这个过程需要蛋白质被添加到新的 突触接触，这反过来又导致mRNA的靶向这些网站， 活动这是学习和记忆的机械基础，因为突触是 通过响应刺激而产生蛋白质来稳定，这些蛋白质对于 它的结构完整性。这种mRNA如何在神经元中调节以产生正确的蛋白质 在正确的地点和时间一直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供资期间开发的工具 来解释mRNA在树突中是如何被调节的。其中一个工具是鼠标， 用荧光标记物标记β-肌动蛋白基因， 活的神经元我们发现mRNA在运输过程中被包裹在一种惰性的形式中 在树突周围。当它靠近受刺激的树突棘时， 它展开它的RNA负载并产生大量蛋白质，但随后又回到休眠状态， 16分钟后，mRNA位于它最后一次被刺激的地方， 小时，等待下一个信号，在那里它将启动另一轮蛋白质。在这 这样，突触接触就建立起来了，与学习和记忆范式一致 依赖于重复性刺激。如果没有进一步的激活信号， 继续它的搜索，移动在短期的连续运动打破了周期， 扩散目前的建议是在发现特定蛋白质后提出的， 与负责将其引导至其位点的mRNA邮政编码结合（邮政编码结合蛋白， ZBP 1）将mRNA锚定在刺激位点。我们构建的模型 这表明mRNA在进一步的刺激下翻译，我们打算把重点放在 通过描述这些事件的动力学和蛋白质， 在这些事件中发挥作用。到目前为止，我们已经研究了β-肌动蛋白mRNA， 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）的调节机制必须 协调许多蛋白质的表达。为此，我们构建了 另一只携带对学习很重要的mRNA的老鼠Arc，已经用一个 不同的荧光标记。在这个提议中，我们将表征β-肌动蛋白的mRNA 和Arc在小鼠中一起，其中两种mRNA都可以通过不同的方法单独检测到。 彩色荧光染料。我们最终提出了CaMK Ⅱ α的第三种混色mRNA， 突触中的一种重要蛋白质。我们的目标是揭示控制 调节不同的mRNA响应于神经元的刺激激活： 它们的合成时机，定位到树突到激活的棘，它们的翻译 抑制，激活和最终降解，以及一些与 这些事件中的每一个。现在，工具已经到位，可以在 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.