Functional analysis of Hu proteins that induce neuronal differentiation

HU蛋白是一种与RNA结合的蛋白，是果蝇ELAV的脊椎动物同源物，在3-非翻译区具有富含AU元素的特定mRNAs的稳定或增强翻译。在这里，使用酵母双杂交系统，我们证明了神经元特异性的HU蛋白可以与自己相互作用。免疫沉淀实验表明，HU蛋白之间的相互作用在哺乳动物细胞中存在，并且在细胞RNA存在的情况下，HU蛋白之间的相互作用被强烈地增强。此外，利用原位化学交联实验，我们发现神经元特异性HU蛋白之一的HUD在细胞中多聚化。交联型HUD多聚体保留了特定的RNA结合能力，并能与额外的HU蛋白相互作用。与此生化特性一致，HUD在两个神经源性细胞系中呈颗粒状分布。这些结果表明，RNA结合形式的HUD协同多聚体形成一种特定的颗粒结构，该结构可能作为含ARE的mRNAs转录后调控的位点。

Hu proteins are RNA-binding proteins that are the vertebrate homologues of Drosophila ELAV, and are implicate in stabilization or enhanced translation of specific mRNAs with AU-rich elements (AREs) in the 3-untranslated region. Here, using the yeast two-hybrid system, we show that neuron-specific Hu proteins can interact with themselves. Immunoprecipitation assays demonstrated that the interaction between Hu proteins occurs in mammalian cells and is strongly enhanced in the presence of cellular RNA, Furthermore, using in situ chemical crosslinking assays, we found that HuD, one of the neuron-specific Hu proteins, multimerizes in cells. The crosslinked HuD multimers retain specific RNA-binding ability and can interact with additional Hu proteins. Consistent with this biochemical property, HuD showed granular distribution in two neurogenic cell lines. These results suggest that the RNA-bound form of HuD multimerizes cooperatively to form a specific granular structure that may serve as a site of post-transcriptional regulation of ARE-containing mRNAs.