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

ELAV样神经元RNA结合蛋白在许多含有神经元的生物体中高度保守，并与神经元的发育和分化有关。哺乳动物神经元特异性ELAV样HU蛋白(Hub、Huc和HUD)和果蝇ELAV蛋白在大鼠PC12细胞中过表达时可诱导突起突起生长，但不能诱导Hur蛋白表达。HUD缺失突变体的功能分析表明，两个保守的RNA结合区(Rbd1和Rbd3)的重要性以及Rbd2和Rbd3之间的连接区对于轴突诱导活性是不可缺少的。进一步的分析表明，连接区一个新的核输出信号(NES)序列介导的HUD核质穿梭对于轴突诱导活性具有重要意义。此外，两个含有连接区的HUD缺失突变体虽然本身没有轴突诱导活性，但它们主要抑制野生型突起的诱导活性，这表明连接区介导的饱和细胞内转运是HUD诱导突起所必需的。有趣的是，相同的显性负性突变体显著抑制了维甲酸诱导的小鼠胚胎癌细胞P19的神经分化。我们的结果表明，在神经元特异性HU蛋白中存在一种新的NES，并且它们通过核质穿梭在细胞质中定位对于启动神经元分化具有重要意义。

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.