Axonal tau mRNA localization coincides with tau protein in living neuronal cells and depends on axonal targeting signal
Axonal tau mRNA localization coincides with tau protein in living neuronal cells and depends on axonal targeting signal
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DOI：
10.1523/jneurosci.21-17-06577.2001
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发表时间：
2001-09-01
影响因子：
5.3
通讯作者：
Ginzburg, I
中科院分区：
文献类型：
--
作者：
Aronov, S;Aranda, G;Ginzburg, I
亚细胞mRNA定位是调节基因表达的基本机制，导致局部蛋白质翻译，从而导致神经元细胞极性的产生。在这项研究中，我们使用了P19胚胎癌细胞，这是适合转染，并选择克隆稳定的细胞系，不过度表达的结构。我们确定了3'非翻译区（3'UTR）tau轴突定位信号，并研究了其对未分化和神经元分化的P19细胞中tau蛋白定位的影响。使用GFP标记的tau蛋白构建体结合原位杂交分析，我们证明了靶向tau蛋白mRNA及其翻译蛋白在轴突和生长锥中的共定位。tau mRNA的3'UTR轴突靶向区域的缺失或突变导致tau mRNA定位的抑制，并且tau mRNA和tau蛋白都保留在细胞体中。3'UTR tau mRNA轴突定位信号和3'UTR MAP 2 mRNA树突靶向信号之间的交换证明蛋白质在轴突或树突中的定位取决于特异性3'UTR靶向信号。此外，轴突中核糖体蛋白的鉴定进一步支持轴突中存在蛋白质合成机制，这是局部翻译的先决条件。因此，建议P19细胞系统可用于分析影响mRNA转运和局部翻译的突变，并且它具有用于检查神经元分化过程的发作的潜力。
Subcellular mRNA localization, a fundamental mechanism for regulating gene expression, leads to local protein translation that results in the generation of neuronal cell polarity. In this study, we have used P19 embryonic carcinoma cells, which are amenable to transfection, and selection of clonal stable cell lines that are not overexpressing the constructs. We identified the 3' untranslated region (3'UTR) tau axonal localization signal and examined its effect on tau protein localization in nondifferentiated and neuronally differentiated P19 cells. Using GFP-tagged tau constructs combined with in situ hybridization analysis, we demonstrated colocalization of the targeted tau mRNA and its translated protein in the axon and growth cone. Absence of or mutation in the 3'UTR axonal targeting region of tau mRNA resulted in suppression of tau mRNA localization, and both tau mRNA and tau protein remained in the cell body. Swapping between the 3'UTR tau mRNA axonal localization signal and the 3'UTR MAP2 mRNA dendritic targeting signal proved that the localization of the proteins into the axon or dendrites depends on the specific 3'UTR targeting signals. Moreover, the identification of ribosomal proteins in the axon lends further support to the presence of protein synthetic machinery in the axons, a prerequisite for local translation. It is suggested therefore that the P19 cell system can be used to analyze mutations that affect mRNA transport and local translation and that it has the potential of being used to examine the onset of the neuronal differentiation process.