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，它们必须具有可塑性和可塑性。能够不断地根据刺激而改变，形成并稳定新的联系。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，这是 mRNA 到这些活性位点的分子基础。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是如何调节该 mRNA 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该提案利用了我们在上一个资助期间开发的工具。为了解决 mRNA 在树突中的调控问题，我们在这一代人中付出了相当大的努力。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，会与荧光结合这些基因表达的单个 mRNA 可以在活细胞中成像并延伸到蛋白质中。我们已仔细验证标记是中性的：它不会改变行为或影响。这些标签之一是针对小鼠记忆形成的，Arc 是一种对大脑做出反应的早期基因。与组成型 β-肌动蛋白不同，神经元刺激对于巩固长期记忆很重要。我们展示的 mRNA 位于最后一次刺激数小时的位置，Arc mRNA 定位仅持续几分钟，并在当前提案报告解决进度后不久就降级。短暂定位的 mRNA 如何影响受刺激刺的长期变化。更重要的是，Arc 会根据单一刺激进行本地化和翻译循环。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”。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，将启动另一轮蛋白质，因为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当前的提议旨在了解翻译热点的动力学，以及它们在可塑性中具有不同作用的不同 mRNA 的空间重叠。 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例如，β-肌动蛋白 mRNA 结合邮政编码结合蛋白 ZBP1 (IMP1)。而 Arc mRNA 则与 FMRP 蛋白结合，进一步的进展将阐明该蛋白。在上一个资助期间，我们又制作了两只小鼠：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：例如，这使我们能够对比神经元处理每个 mRNA 的方式。在其本地化和翻译过程中，CaMKII 取得了意想不到的成果。 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它们各自的 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。