Mechanism of mRNA Localization and Localized Translation in Neurons

神经元中mRNA定位和定位翻译的机制

• 批准号: 10586910
• 负责人: Sulagna Das
• 金额: $ 66.58万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-03 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586910
• 关键词: 3' Untranslated Regions; Actins; Address; Affect; Antibodies; Behavior; Binding; Binding Proteins; Brain; Caring; Cell physiology; Cells; Color; Complex; Cytoplasmic Granules; Dendrites; Environment; Epitopes; Event; FMR1; Fluorochrome; Funding; Generations; Genes; Genetic Transcription; Genetic Translation; Genetically Engineered Mouse; Heart; Hour; Hybrids; Image; Immediate-Early Genes; Individual; Instruction; Investigation; Kinetics; Knock-in; Label; Learning; Location; Memory; Messenger RNA; Methodology; Microscopy; Modeling; Molecular; Mus; Neurons; Outcome; Peptide Synthesis; Physiological; Play; Process; Production; Protein Biosynthesis; Proteins; RNA; RNA Editing; Reagent; Regulation; Reporting; Resolution; Role; Signal Transduction; Site; Slice; Spatial Distribution; Spottings; Stimulus; Structural Protein; Synapses; Technology; Time; Tissues; Transgenic Animals; Translating; Translations; Travel; Vertebral column; Work; base; beta Actin; brain tissue; calmodulin-dependent protein kinase II; follow-up; image translation; imaging system; innovation; long term memory; mRNA tagging; malignant neurologic neoplasms; nervous system disorder; novel; protein activation; real-time images; response; spatiotemporal; stem; synaptogenesis; temporal measurement; tool

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, as we have shown in our previous work. This is the molecular basis of learning and memory since the synapse is stabilized by the production of proteins in response to stimulation that is important for its function and 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 developed during the last funding period to address how mRNA is regulated in dendrites. We have expended considerable effort in the generation of genetically engineered mice wherein the loci of three neuronally expressed genes important for learning and memory have been tagged with stem loops that, when expressed in the mRNA bind to fluorescent proteins. The single mRNAs expressed from these genes can be imaged in living cells and extended into live tissues. We have taken care to verify that the tagging is neutral: it does not alter the behavior or affect memory formation in the mice. One of these tags is for Arc, an immediate early gene in response to neuronal stimulation important for consolidating long term memory. Unlike the constitutive -actin mRNA, which we showed sits at the place where it was last stimulated for hours, Arc mRNA localizes only for a few minutes, and degrades soon after. The current proposal reports on the progress to solving how transiently localized mRNAs can impact long term changes at the stimulated spines. The surprise was that Arc undergoes cycles of localization and translation in response to a single stimulus. Even more incredible is that the translation occurs spatially at the same spot, so the mRNA in the next cycle finds the site of previous localization and builds up a continuous “hotspot” of localized protein synthesis. This in contrast to the β-actin mRNA, which persists at the stimulated site, awaiting the next signal, wherein it will initiate another round of proteins. Because β-actin is a structural protein, the synaptic contact is built up with rounds of translation, consistent with a learning and memory paradigm that relies on repetitive stimulation. The current proposal is directed towards understanding the kinetics of translation hotspots, and their spatial overlap for different mRNAs with distinct roles in plasticity. We crossed the β-actin and Arc mice to homozygosity where both mRNAs were individually detectable by different colored fluorochromes in the same neuron. We have learned from this mouse that the two mRNAs were handled differently by the neuron, and traveled in independent “granules”, likely resulting from differences in their associated proteins. For instance, β-actin mRNA bound the zipcode binding protein, ZBP1 (IMP1) whereas Arc mRNA instead bound the protein FMRP. Further progress will elucidate the protein composition of each granule in more detail. We have made two more mice during the last funding period: a GCN4 epitope tagged (“Suntag”) Arc mouse that will allow us to see the translation sites of Arc protein using a fluorescent single chain antibody in living cells and tissues (we developed this tag previously), and a CaMKII mouse where the mRNA is distinguishable from either β-actin mRNA or Arc mRNA by hybrid fluorescent tags. This allows us now to contrast how the neuron handles each mRNA, for example in its localization and translation. Over the last funding period, an unexpected result was that the CaMKII mRNA localized in the spines, unlike either Arc or β-actin mRNA, that stayed at the base of the spines. This indicated that subtleties in the localization of these mRNAs may underlie a physiological purpose. We intend to investigate this by determining the sequences that likely direct this mRNA into the spines. Ultimately, we intend to find the proteins bound specifically to these mRNAs and how they might affect the regulation of their respective mRNAs. This will use the technologies of RNA editing and proximity labeling, which will allow us to interrogate the associated RNAs and proteins that make each species of mRNA granule unique.

抽象的 神经元是大脑的基本细胞单位。为了使神经元正常工作，它们必须是可塑的并且 能够不断地根据刺激而改变，形成并稳定新的联系。这 该过程需要将蛋白质添加到新的突触接触中，而这又是靶向的结果 正如我们在之前的工作中所展示的，这就是分子基础 学习和记忆，因为突触通过响应于响应而产生蛋白质来稳定 对其功能和结构完整性很重要的刺激。这个 mRNA 是如何被调控的 神经元在正确的地点和时间制造正确的蛋白质一直是我们研究的主题 多年来这笔资金。该提案利用了我们在上一个资助期间开发的工具 解决树突中 mRNA 的调控问题。我们这一代人付出了巨大的努力 基因工程小鼠的三个神经元表达基因的位点对学习很重要 和记忆已被标记为茎环，当在 mRNA 中表达时，会与荧光结合 蛋白质。这些基因表达的单个 mRNA 可以在活细胞中成像并扩展到 活组织。我们已仔细验证标签是中性的：它不会改变行为或影响 小鼠的记忆形成。其中一个标签是针对 Arc 的，它是一种立即响应的早期基因 神经元刺激对于巩固长期记忆很重要。与组成型 -肌动蛋白不同 我们展示的 mRNA 位于最后一次刺激数小时的位置，Arc mRNA 定位 只持续几分钟，很快就会降解。目前的提案报告了解决问题的进展情况 短暂定位的 mRNA 如何影响受刺激脊柱的长期变化。惊喜 Arc 会根据单一刺激进行本地化和翻译循环。更 令人难以置信的是，翻译在空间上发生在同一位置，因此下一个循环中的 mRNA 会找到 先前定位的位点，并建立了局部蛋白质合成的连续“热点”。这在 与 β-肌动蛋白 mRNA 不同，β-肌动蛋白 mRNA 持续存在于刺激位点，等待下一个信号，其中 将启动另一轮蛋白质。由于 β-肌动蛋白是一种结构蛋白，因此建立了突触接触 进行多轮翻译，符合依赖重复的学习和记忆范式 刺激。当前的提议旨在了解翻译热点的动力学， 以及在可塑性中具有不同作用的不同 mRNA 的空间重叠。我们穿过 β-肌动蛋白 Arc 小鼠达到纯合性，其中两种 mRNA 均可通过不同颜色单独检测到 同一神经元中的荧光染料。我们从这只小鼠身上了解到，这两种 mRNA 都被处理过 神经元以不同的方式移动，并以独立的“颗粒”的形式传播，这可能是由于它们的差异造成的 相关蛋白质。例如，β-肌动蛋白 mRNA 结合邮政编码结合蛋白 ZBP1 (IMP1) 而 Arc mRNA 则结合了 FMRP 蛋白。进一步的进展将阐明该蛋白质 更详细地了解每个颗粒的成分。在上一次资助期间，我们又制作了两只老鼠： 带有 GCN4 表位标记（“Suntag”）的 Arc 小鼠，使我们能够看到 Arc 蛋白的翻译位点 在活细胞和组织中使用荧光单链抗体（我们之前开发了这个标签）， 和 CaMKII 小鼠，其中 mRNA 可通过以下方式与 β-肌动蛋白 mRNA 或 Arc mRNA 区分开： 混合荧光标签。这让我们现在可以对比神经元如何处理每个 mRNA，例如 其本地化和翻译。在上一个资助期间，一个意想不到的结果是 CaMKII mRNA 位于棘中，与 Arc 或 β-肌动蛋白 mRNA 不同，后者位于棘的基部。 这表明这些 mRNA 定位的微妙之处可能是其生理目的的基础。 我们打算通过确定可能引导该 mRNA 进入刺的序列来研究这一点。 最终，我们打算找到与这些 mRNA 特异性结合的蛋白质以及它们如何影响 各自mRNA的调节。这将使用 RNA 编辑和邻近技术 标记，这将使我们能够询问相关的 RNA 和蛋白质，这些 RNA 和蛋白质使每个物种 mRNA颗粒独特。