Mechanism of mRNA Localization and Localized Translation in Neurons

神经元中 mRNA 定位和定位翻译的机制

基本信息

批准号：
10708979
负责人：
Sulagna Das
金额：
$ 67.63万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
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/10708979
关键词：
3&apos Untranslated Regions Actins Address Affect Antibodies Behavior Binding Binding Proteins Brain Breeding Caring Cell physiology Cells Color Complex Cytoplasmic Granules Dendrites Environment Epitopes Event FMR1 Fluorochrome Funding Generations Genes Genetic Transcription Genetically Engineered Mouse Heart Hour Hybrids Image Immediate-Early Genes Individual 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 Visualization Work base beta Actin brain tissue follow-up image translation imaging system innovation long term memory mRNA Translation mRNA tagging malignant neurologic neoplasms nervous system disorder novel protein activation real-time images response spatiotemporal stem synaptogenesis temporal measurement tool

项目摘要

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，它们必须是塑料，并且不断能够响应刺激，形成和稳定新连接而改变。这过程需要将蛋白质添加到新的突触接触中，这反过正如我们在以前的工作中所表明的那样，这些活动位置的mRNA。这是分子基础学习和记忆，因为突触通过蛋白质的产生而稳定刺激对其功能和结构完整性很重要。如何调节此mRNA 神经元在正确的位置和时间使正确的蛋白质成为我们调查的主题多年来，这笔资金。该提案利用了我们在上一个资金期间开发的工具解决在树突中如何调节mRNA。我们已经探索了这一代人的巨大努力在基因工程的小鼠中，其中三个神经元表达基因的局部对于学习很重要和记忆已用茎回路标记，当mRNA与荧光结合时，蛋白质。从这些基因表达的单个mRNA可以在活细胞中成像，并延伸到活组织。我们已经注意到标签是中性的：它不会改变行为或影响小鼠的记忆形成。这些标签之一是用于ARC，这是一个直接的早期基因神经元刺激对于整合长期记忆很重要。与本构-actin不同 mRNA，我们展示的位于最后一次刺激数小时的地方，弧mRNA定位只有几分钟，不久之后就会退化。当前的提案报告了解决的进度瞬时局部mRNA如何影响刺激的刺处的长期变化。惊喜是响应单个刺激而经历定位和翻译的周期。更令人难以置信的是，翻译在空间上发生在同一位置，因此下一个周期中的mRNA发现先前定位的位点，并建立了局部蛋白质合成的连续“热点”。这是与β-肌动蛋白mRNA形成鲜明对比，该mRNA持续存在于刺激位点，等待下一个信号，其中它将引发另一轮蛋白质。由于β-肌动蛋白是一种结构蛋白，因此建立了突触接触与翻译的回合，与依赖重复性的学习和记忆范式一致刺激。当前的建议是针对理解翻译热点的动力学的他们的空间重叠在不同的mRNA中，在可塑性中具有不同的作用。我们越过β-肌动蛋白，弧小鼠到纯合性，在不同的颜色中可以单独检测两个mRNA 同一神经元中的荧光染料。我们从这只鼠标中学到了两个mRNA。神经元有所不同，并以独立的“颗粒”行进，可能是由于其差异而产生的相关蛋白质。例如，β-肌动蛋白mRNA结合了Zipcode结合蛋白ZBP1（IMP1）而弧mRNA代替结合蛋白质FMRP。进一步的进展将阐明蛋白质每个颗粒的组成更详细。在最后一个资金期间，我们又养了两只小鼠： gcn4情节标记为（“ suntag”）弧小鼠，它将使我们能够看到ARC蛋白的翻译位点在活细胞和组织中使用荧光单链抗体（我们以前开发了此标签），和camkii小鼠，其中mRNA可与β-肌动蛋白mRNA或弧mRNA区分开混合荧光标签。例如，这使我们现在可以对比神经元如何处理每个mRNA，例如在其本地化和翻译中。在最后一个资金期间，一个意外的结果是Camkii 与静脉或β-肌动蛋白mRNA不同的mRNA定位在棘突中，该mRNA留在脊柱的底部。这表明这些mRNA定位的微妙之处可能是物理目的的基础。我们打算通过确定可能将此mRNA引导到脊柱的序列来研究这一点。最终，我们打算找到专门绑定到这些mRNA的蛋白质以及它们可能如何影响他们各自的mRNA的调节。这将使用RNA编辑和接近性的技术标签，这将使我们能够询问相关的RNA和蛋白质，使每种物种的每种物种 mRNA颗粒独特。