哺乳類脳神経幹細胞の特性の経時変化を制御するエピゲノム修飾と細胞間シグナルの研究

在过去的一年里，吴继续分析Fibrillarin在神经干细胞（NSCs）时间命运改变中的功能。结果，Wu发现在增殖阶段，RNA干扰介导的Fibrillarin基因敲低至约50%，可促进神经干细胞的过早神经元分化。因此，Notch信号传导的活性是神经干细胞自我更新的重要因素，在敲低Fibrillarin后，Notch信号传导的活性显著降低。相反，原纤蛋白的过表达增加了神经干细胞的数量。这些结果表明，Fibrillarin是神经干细胞增殖所必需的。接下来，Wu试图揭示Fibrillarin影响NSC命运的机制。由于Fibrillarin是rRNA的甲基转移酶，rRNA的甲基化水平可能会影响翻译，Wu询问Fibrillarin的敲除是否影响整体蛋白质翻译或仅影响特定靶标。使用化学探针追踪新合成的蛋白质，Wu证实了RNAi介导的Fibrillarin敲低至约50%并不影响整体蛋白质翻译。这些结果表明Fibrillarin通过优先翻译靶mRNA来调节细胞命运。然后，为了寻找Fibrillarin的靶点，Wu与Iwasaki Shitano博士合作，进行了核糖体分析，这是一种基于深度测序检测翻译mRNA的新方法。即使这个实验还在进行中，我认为这个实验的结果将有助于我们更好地了解NSC的命运是如何精确控制的。在完成JSPS奖学金后，吴将继续在我的实验室完成这个项目。

In the last year, Wu continued to analyze the function of Fibrillarin in temporal fate change of neural stem cells (NSCs). As a result, Wu found that RNAi-mediated knockdown of Fibrillarin to ~50% at proliferative stage promoted premature neuronal differentiation of NSCs. Consistently, the activity of Notch signaling which is important for self-renewal of NSCs was strongly reduced after knockdown of Fibrillarin. In contrast, overexpression of Fibrillarin increased the population of NSCs. These results suggest that Fibrillarin is essential for proliferation of NSCs. Next, Wu tried to uncover the mechanism by which Fibrillarin affects the fate of NSCs. Because Fibrillarin is a methyltransferase of rRNA and methylation level of rRNA may affect translation, Wu asked that whether knockdown of Fibrillarin affects global protein translation or only impacts on specific targets. Using chemical probe to trace newly synthesized protein, Wu confirmed that RNAi-mediated knockdown of Fibrillarin to ~50% did not affect global protein translation. These results imply that Fibrillarin regulates cell fate through preferentially translating of target mRNAs. Then, to search the targets of Fibrillarin, Wu, cooperating with Dr. Iwasaki Shitano, performed ribosome profiling, a novel method to detect translating mRNAs based on deep-sequencing. Even this experiment is still ongoing, I think that results of this experiment will help us to better understand how NSC fate is precisely controlled. After finish of JSPS fellowship, Wu will continue to complete this project in my lab.