The Role of RNA Binding Protein Hu in Neuronal Differentiation

神经元Hu RNA结合蛋白在神经元祖细胞退出细胞周期后立即表达，并在有丝分裂后的神经元中继续表达。最近的研究表明，Hu的过表达足以通过与哺乳动物神经系统中的几种靶mRNA的UTR区域结合来诱导神经元分化。揭示神经元分化时间调控的分子机制。通过Hu的转录后调节，我们鉴定了Hu相关蛋白。我们从感染Hu-腺病毒的培养细胞提取物中分离了与Hu相关的RNP复合物，并通过MALDI-TOF Mass鉴定了三种蛋白。我们发现hnRNPK是一种RNA结合蛋白，它是Hu相关蛋白之一，在体外和体内与Hu的靶基因p21 mRNA 3 'UTR结合，并抑制其在非神经元和神经元细胞中的翻译。最近，有报道称p21不仅抑制细胞周期，而且作为发育调节剂诱导新生神经元的轴突和树突。推测Hu和hnRNPK这两种RNA结合蛋白可能通过转录后调节p21 mRNA来调控神经元的分化。通过报告基因分析，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 'UTR。此外，我们发现，胡诱导神经突生长，停止细胞周期和上调p21蛋白的产生在小鼠神经母细胞瘤N1 E-115细胞。这些作用被hnRNPK部分抑制。综上所述，这些数据表明Hu和hnRNPK通过调节下游基因来控制神经元分化的时间。

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.