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.

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，它们必须是可塑性的 能够不断地改变对刺激的反应，形成和稳定新的联系。这 这一过程需要将蛋白质添加到新的突触接触中，这反过来又是靶向的结果 信使核糖核酸到这些活性部位，正如我们在以前的工作中所显示的那样。这就是分子基础 学习和记忆，因为突触是通过产生蛋白质来稳定的 对其功能和结构完整性很重要的刺激。这种信使核糖核酸是如何在 神经元在正确的时间和地点制造正确的蛋白质一直是我们研究的主题 在这笔资金的这些年里。这项提案利用了我们在上一个资助期开发的工具 来解决树突中的mRNA是如何被调控的。我们在这一代人中付出了相当大的努力 其中三个神经元表达对学习重要的基因的基因工程小鼠 和记忆已经被标记了茎环，当在mRNA中表达时，这些茎环与荧光结合 蛋白质。从这些基因表达的单个mRNAs可以在活细胞中成像并延伸到 活的纸巾。我们已经小心地验证了标记是中性的：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，它是一种直接早期的基因，对 神经元刺激对巩固长期记忆很重要。与构成-肌动蛋白不同 我们发现它位于最后一次被刺激的地方几个小时，Arc mRNA定位 只有几分钟，很快就会降解。目前的提案报告了解决方案的进展情况 短暂局部化的mRNAs如何影响刺激脊椎的长期变化。令人惊讶的是 Arc经历了本地化和翻译的循环，以回应单一的刺激。甚至更多 令人难以置信的是，翻译在空间上发生在同一地点，所以下一个周期中的mRNA找到了 并建立了一个持续的定位蛋白质合成的“热点”。此入站 与β-肌动蛋白mRNA形成鲜明对比的是，它持续存在于刺激部位，等待下一个信号，其中它 将启动另一轮蛋白质。由于β-肌动蛋白是一种结构蛋白，因此建立了突触联系 经过几轮翻译，与依赖重复的学习和记忆范式保持一致 刺激。目前的建议旨在了解翻译热点的动力学， 以及它们在可塑性中扮演不同角色的不同mRNAs的空间重叠。我们穿过了β-肌动蛋白 使小鼠达到纯合子状态，其中两种mRNAs可被不同颜色分别检测到 同一个神经元中的荧光染料。我们从这只老鼠那里了解到，这两个mRNA是被处理过的 不同的神经元，并在独立的“颗粒”中旅行，可能是由于它们的不同 相关蛋白质。例如，β-肌动蛋白mRNA结合了邮编结合蛋白ZBP1(IMP1)。 而Arc mRNA则与FMRP蛋白结合。进一步的进展将阐明这种蛋白质。 更详细地说明每个颗粒的组成。在上个资助期，我们又制造了两只老鼠： 一个Gcn4表位标记(“Suntag”)Arc小鼠，将使我们能够看到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)， 以及CaMKII小鼠，其中该基因可通过以下方式与β-肌动蛋白基因或Arc基因区分开来 混合荧光标签。例如，这使我们能够对比神经元如何处理每一个mRNA 在本地化和翻译方面。在上一个资助期，一个意想不到的结果是，CAMKII 与Arc或β-Actin mRNA不同，它定位于脊椎，停留在脊椎的底部。 这表明，这些mRNAs定位的微妙之处可能是生理目的的基础。 我们打算通过确定可能将该mRNA引导到脊椎中的序列来研究这一点。 最终，我们打算找到与这些mrna特异结合的蛋白质以及它们可能如何影响。 它们各自的mRNAs的调控。这将使用RNA编辑和邻近技术 标记，这将允许我们询问相关的RNA和蛋白质，这些RNA和蛋白质构成了每一种 信使核糖核酸颗粒独特。