Cytoplasmic localization is required for the mammalian ELAV-like protein HuD to induce neuronal differentiation.

ELAV-like neuronal RNA-binding proteins are highly conserved in many neurone-containing organisms and have been implicated in neuronal development and differentiation. Mammalian neurone-specific ELAV-like Hu proteins (HuB, HuC and HuD) and Drosophila ELAV, but not HuR, were found to induce neurite outgrowth when over-expressed 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 neurone-specific Hu proteins and the importance of their cytoplasmic localization, through nucleocytoplasmic shuttling, for the initiation of neuronal differentiation.