Mechanism of Actin mRNA Localization and Localized Translation in Neurons

神经元中肌动蛋白 mRNA 定位和定位翻译的机制

• 批准号: 9127383
• 负责人: Robert H Singer
• 金额: $ 52.16万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9127383
• 关键词: 3' Untranslated Regions; Actins; Address; Adhesions; Affect; Affinity; Behavior; Binding; Binding Proteins; Biological Assay; Birth; Brain; Brain-Derived Neurotrophic Factor; Breast cancer metastasis; Cell Line; Cell physiology; Cells; Cellular Structures; Characteristics; Chimeric Proteins; Cytoplasmic Granules; Defect; Dendrites; Destinations; Detection; Development; Diabetes Mellitus; Disease; Distant; Drug Addiction; Drug resistance; Embryo; Event; Fibroblasts; Fluorescence Resonance Energy Transfer; Frequencies; Funding; Generations; Genetic Translation; Goals; Grant; Growth; Growth Cones; Heart; Hour; Image; Immunofluorescence Immunologic; Individual; Kinetics; Knockout Mice; Knowledge; Label; Lead; Learning; Life; Location; Maintenance; Mammary gland; Maps; Measures; Memory; Messenger RNA; Metabolism; Methodology; Methods; Microscopy; Movement; Mus; Neurites; Neurons; Neurotransmitter Receptor; Newborn Infant; Open Reading Frames; Phosphotransferases; Photons; Potassium Chloride; Protein Biosynthesis; Protein Family; Proteins; RNA; Reading; Regulation; Reporter; Resistance; Ribosomes; Role; Shapes; Signal Transduction; Site; Stimulus; Structure; Subfamily lentivirinae; Synapses; Talin; Techniques; Technology; Time; Tissues; Trans-Activators; Transgenic Animals; Transgenic Mice; Translating; Translational Regulation; Translations; Travel; Vertebral column; aptamer; base; cell growth; cell motility; design; disorder prevention; innovation; knock-down; malignant neurologic neoplasms; meter; method development; mouse model; nervous system disorder; neurodevelopment; neuronal cell body; new technology; novel; polypeptide; postsynaptic; prevent; public health relevance; research study; single molecule; small hairpin RNA; tool; translation assay

DESCRIPTION (provided by applicant): ABSTRACT: The regulation of mRNA is critical to cellular function. Over the years of this grant we discovered that an important mode of regulation is the ability of messenger RNA to become localized at specific regions in cells where synthesis of specific proteins can be spatially compartmentalized. This has profound implications for cell structure and function since all cells have characteristic shapes and structure, which are essential for them to perform their function. The best example of this is the neuron, where the synapses are very far away from the cell body, sometimes meters away. Messenger RNA may have to travel these far distances and then be activated to make proteins upon stimulation of specific synapses at a precise moment. This is the basis of learning and memory. The mechanism by which the mRNA can remain quiescent for long periods of time and then become active upon a specifically localized stimulus is unknown. The Specific Aims of this proposal are directed toward developing new technology to address this question. We have made significant experimental, technical and conceptual advances that have allowed us to observe single molecules of mRNA. For instance we have made a mouse where every molecule of the ss-actin mRNA, which makes an essential protein is labeled. This will allow us to observe these molecules in living cells and tissues. We found that the mRNA travels to distant regions of the cell because of a sequence known as the zipcode. We discovered that this sequence binds a protein, the zipcode binding protein (ZBP1) and this binding silences the mRNA until it reaches its final destination. To activate the mRNA to translate, the protein must be modified at its destination by a kinase that phosphorylates it at a specific site. We believe ZBP1 is the key to understanding the regulatory events that occur at specific locations in the cell, for instance at the synapse, where ss-actin is necessary for stabilizing spines important for their presentation to incoming signals. In support of the importance of this protein, if we delete it in mice, the result is lethal, newborn mice do not survive and their brains show defects in organization of the neuronal layers. We have shown that the protein is essential for proper migration of cells, such as fibroblasts and neurons, and this we believe is due to the ability of the cell to direct the synthesis of actin in a polarized location, where it can polymerize and drive the extension of cell structures involved in movement. ZBP1 is also implicated in disease prevention. After birth, the expression of ZBP1 is repressed, but we have been able to make a transgenic mouse that expresses ZBP1 exogenously in the brain and show that these mice have profoundly altered behavior: they become resistant to drug addiction. Furthermore, expression of ZBP1 in the mammary gland makes the mice resistant to breast cancer metastasis. We have also recently discovered that the ZBP1 family of proteins (there are three) is also implicated in preventing neurological diseases and diabetes. Hence not only will study of this protein reveal how mRNA is regulated, but also how disruption of this regulation can lead to a broad variety of diseases.

描述(由申请人提供)： 摘要：信使核糖核酸的调控对细胞功能至关重要。多年来，我们发现一种重要的调控模式是信使RNA能够定位于细胞中特定的区域，在那里特定蛋白质的合成可以在空间上被分隔。这对细胞的结构和功能有深远的影响，因为所有的细胞都有其特有的形状和结构，这是它们履行其功能所必需的。最好的例子是神经元，那里的突触离细胞体很远，有时甚至几米远。信使RNA可能需要走这么远的距离，然后在准确的时刻被激活，在刺激特定突触时制造蛋白质。这是学习和记忆的基础。信使核糖核酸能够长时间保持静止，然后在特定的局部刺激下变得活跃的机制尚不清楚。这项提案的具体目标是开发新技术来解决这一问题。我们已经取得了重大的实验、技术和概念上的进步，使我们能够观察到单分子的mRNA。例如，我们已经制造了一只小鼠，其中每一个分子的ss-肌动蛋白mRNA都被标记，而ss-肌动蛋白mRNA是一种必不可少的蛋白质。这将使我们能够观察活细胞和组织中的这些分子。我们发现，由于一种被称为邮政编码的序列，信使核糖核酸转移到细胞的较远区域。我们发现，这个序列与一种蛋白质--邮政编码结合蛋白(ZBP1)结合，这种结合会使mRNA沉默，直到它到达最终目的地。为了激活要翻译的信使核糖核酸，蛋白质必须在其目的地被一种在特定位置磷酸化它的蛋白所修饰。我们认为，ZBP1是理解发生在细胞特定位置的调控事件的关键，例如在突触，在突触中，ss-肌动蛋白是稳定脊椎所必需的，而脊椎对传入信号的呈现至关重要。为了支持这种蛋白质的重要性，如果我们在小鼠身上删除它，结果是致命的，新生的小鼠无法存活，它们的大脑显示出神经层组织的缺陷。我们已经证明，该蛋白对于成纤维细胞和神经元等细胞的正常迁移是必不可少的，我们认为这是由于细胞能够在极化位置指导肌动蛋白的合成，在那里它可以聚合并驱动参与运动的细胞结构的延伸。ZBP1还与疾病预防有关。出生后，ZBP1的表达受到抑制，但我们已经能够制造出在大脑中外源表达ZBP1的转基因小鼠，并表明这些小鼠已经深刻地改变了行为：它们对药物成瘾产生了抗药性。此外，ZBP1在乳腺中的表达使小鼠对乳腺癌转移具有抵抗力。我们最近还发现，ZBP1蛋白家族(有三个)也与预防神经疾病和糖尿病有关。因此，对这种蛋白质的研究不仅将揭示mRNA是如何调控的，而且还将揭示这种调控的破坏如何导致多种疾病。