The Role of RNA Binding Protein Hu in Neuronal Differentiation

神经元HuRNA结合蛋白在神经前体退出细胞周期后立即表达，并在有丝分裂后神经元中继续表达。最近的研究表明，在哺乳动物神经系统中，HU的过度表达足以通过与几个靶mRNAs的非编码区结合来诱导神经元分化。揭示神经分化时机调控的分子机制。通过HU转录后调控，我们鉴定了HU相关蛋白。我们从HU-腺病毒感染的培养细胞提取液中分离出与HU相关的RNP复合体，并用MALDI-TOF MS鉴定了三种蛋白质。我们揭示了HU结合蛋白之一的RNA结合蛋白hnRNPK在体外和体内都能与p21mRNA3‘UTR结合，并抑制其在非神经细胞和神经细胞中的翻译。最近的研究表明，p21不仅可以抑制细胞周期，还可以作为发育调节剂诱导新生神经元中的轴突和树突。我们推测，神经元的分化是由两种RNA结合蛋白HU和hnRNPK通过转录后调控p21mRNA来调节的。通过报告实验，我们发现在神经元分化过程中，p21的表达受到转录后调控，这种调控依赖于其3‘端非编码区。此外，我们还发现，HU可诱导小鼠神经母细胞瘤N1E-115细胞突起生长，使细胞周期停止，并上调p21蛋白的表达。HnRNPK可部分抑制上述作用。综上所述，这些数据表明，Hu和hnRNPK通过调控下游基因来控制神经元分化的时机。

Neuronal Hu RNA binding proteins are expressed immediately after the neuronal progenitor withdraw from cell cycle and continues to be expressed in postmitotic neurons. Recent studies indicate that overexpression of Hu is sufficient to induce neuronal differentiation by binding to UTR region of several target mRNAs in mammalian nervous system. To reveal molecular mechanism underlining timing-control of neuronal differentiation through. posttranscriptional regulation by Hu, we identified Hu associating proteins. We isolated RNP complexes associated with Hu from extracts of Hu-adenovirus infected culture cells and three proteins were identified by MALDI-TOF Mass. We revealed that hnRNPK, an RNA binding protein which is one of Hu associating proteins, bound to p21 mRNA 3'UTR, known as a target gene of Hu in vitro and in vivo, and repressed its translation in both nonneuronal and neuronal cells. Recently, it was reported that p21 not only inhibits the cell cycle but also contributes as a developmental regulator that induces axons and dendrites in newborn neurons. Then we speculated that the neuronal differentiation was regulated by two RNA binding proteins, Hu and hnRNPK through post-transcriptional regulation of p21 mRNA. By reporter assay, we found that p21 expression is controlled posttranscriptionally during neuronal differentiation and this regulation was dependent on its 3'UTR. Furthermore we showed that Hu induced neurite-outgrowth, stopped the cell cycle and upregulated p21 protein production in the mouse neuroblastoma N1E-115 cells. These effects were partially inhibited by hnRNPK. Taken all together, these data would suggest that Hu and hnRNPK controlled the timing of neuronal differentiation by regulating downstream genes.