权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

摘要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，能够不断地对刺激做出反应，形成和稳定新的连接。这这一过程需要蛋白质被添加到新的突触接触中，而这反过来又是靶向的结果。这些活动位点的mRNA，正如我们在我们以前的工作中所示。这是分子基础，学习和记忆，因为突触是通过响应蛋白质的产生来稳定的。刺激，这对它的功能和结构完整性很重要。这种mRNA是如何被调节的，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在这些年的资助中。该提案利用了我们在上一个供资期开发的工具来解释mRNA在树突中是如何被调节的。我们花了相当大的努力，在基因工程小鼠中，三个神经元表达的基因的位点对学习很重要，和记忆都被标记了茎环，当表达在mRNA中时， proteins.从这些基因表达的单个mRNA可以在活细胞中成像并延伸到细胞中。活组织我们已经仔细验证了标记是中性的：它不会改变行为或影响老鼠的记忆形成其中一个标签是Arc，这是一个响应于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显示，Arc mRNA位于最后一次刺激的位置数小时，Arc mRNA定位于几分钟后就会降解目前的提案报告了解决瞬时定位的mRNA如何影响受刺激脊髓的长期变化。的惊喜 Arc对单一刺激的反应是经历定位和翻译的循环。更令人难以置信的是，翻译在空间上发生在同一个地方，所以mRNA在下一个循环中找到了在此基础上，构建了定位蛋白质合成的连续“热点”。这与β-肌动蛋白mRNA相反，其持续存在于刺激位点，等待下一个信号，其中它会引发新一轮的蛋白质合成因为β-肌动蛋白是一种结构蛋白，随着翻译的循环，与依赖于重复的学习和记忆范式相一致，刺激.目前的建议是针对理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。我们穿过β-肌动蛋白，将Arc小鼠转化为纯合性，其中两种mRNA都可以通过不同颜色的荧光标记单独检测到。荧光染料在同一个神经元。我们从这只老鼠身上了解到，不同的神经元，并在独立的“颗粒”，可能是由于他们的差异，相关蛋白质例如，β-肌动蛋白mRNA结合zipcode结合蛋白ZBP 1（IMP 1）而Arc mRNA则与蛋白质FMRP结合。进一步的进展将阐明蛋白质更详细地描述每个颗粒的组成。我们在上一个资助期内又制造了两只老鼠： GCN 4表位标记的（“Suntag”）Arc小鼠，其将允许我们看到Arc蛋白的翻译位点在活细胞和组织中使用荧光单链抗体（我们以前开发了这种标签），和CaMKIImRNA小鼠，其中mRNA通过以下方式与β-肌动蛋白mRNA或Arc mRNA区分开：杂交荧光标记。这使我们现在可以对比神经元如何处理每一种mRNA，例如，在本地化和翻译方面。在上一个融资期，一个意想不到的结果是，CaMKII mRNA位于棘中，不像Arc或β-肌动蛋白mRNA，其停留在棘的基部。这表明这些mRNA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引导这种mRNA进入棘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编辑和邻近技术标签，这将使我们能够询问相关的RNA和蛋白质，使每个物种的 mRNA颗粒独特。