Studies on the Biological Functions of Neuron-specific RNA-binding Proteins in Cultured Cells

elav样神经元rna结合蛋白在许多含神经元生物中高度保守，并与神经元发育和分化有关。哺乳动物神经元特异性ELAV样Hu蛋白（HuB， HuC和HuD）和果蝇ELAV（但不包括HuR）在大鼠PC12细胞中过表达时可诱导神经突生长。HuD缺失突变体的功能分析表明，两个保守的rna结合结构域（RBDs 1和3）和RBDs 2和3之间的连接区域对于神经突诱导活性是不可或缺的。进一步的分析表明，由连接区一个新的核输出信号（NES）序列介导的HuD核胞质穿梭对神经突诱导活性的重要性。此外，两个含有连接体区域的HuD缺失突变体主要抑制野生型神经突诱导活性，尽管它们本身没有神经突诱导活性，这表明由连接体区域介导的饱和细胞内运输是HuD诱导神经突的必要条件。有趣的是，相同的显性阴性突变体显著抑制维甲酸诱导的小鼠胚胎癌P19细胞的神经元分化。我们的研究结果表明，在神经元特异性的Hu蛋白中存在一种新的NES，以及它们的细胞质定位（通过核胞质穿梭）对神经元分化的启动具有重要意义。

ELAV-like neuronal RNA-binding proteins are highly conserved in many neuron-containing organisms and have been implicated in neuronal development and differentiation. Mammalian neuron-specific ELAV-like Hu proteins (HuB, HuC and HuD) and Drosophila ELAV, but not HuR, were found to induce neurite outgrowth when overexpressed in rat PC12 cells. Functional analysis of HuD deletion mutants demonstrated the importance of two conserved RNA-binding domains (RBDs 1 and 3) and the indispensability of the linker region between RBDs 2 and 3 for the neurite-inducing activity. Further analyses suggested the importance of nucleocytoplasmic shuttling of HuD mediated by a novel nuclear export signal (NES) sequence in the linker region for the neurite-inducing activity. Moreover, two HuD deletion mutants containing the linker region dominantly inhibited the wild-type neurite-inducing activity, although they had no neurite-inducing activity per se, suggesting that saturable intracellular trafficking mediated by the linker region is required for the neurite induction by HuD. Interestingly, the same dominant negative mutants significantly inhibited retinoic acid-induced neuronal differentiation of mouse embryonal carcinoma P19 cells. Our results suggest the presence of a novel NES in neuron-specific Hu proteins and the importance of their cytoplasmic localization, through nucleocytoplasmic shuttling, for the initiation of neuronal differentiation.