Analysis of multifunctional property of Y-box protein in the nervous system.

Y-box蛋白在神经系统中的多功能特性分析。

基本信息

批准号：
15500268
负责人：
KOBAYASHI Shunsuke
金额：
$ 2.05万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

In order to investigate functions of a Y-box protein rBYB1 in cytoplasm and nucleus of the neuronal cells, we used rat brain extract, primary hippocampal neurons and nucleus of neuroblastoma NG108-15 cells. rBYB1 was found to be considerably expressed in the cytoplasm of pre- and early postnatal brains, and then decreased to adult levels with brain development. Distribution of the rBYB1 in a sucrose density gradient revealed that the protein interacts with polysomes via RNA. It has been reported that Y-box proteins bind to mRNAs and modulate translation. We isolated mRNAs interacting with rBYB1 in the developing brain by immunoprecipitation using anti-rBYB1 antibody and identified some mRNAs encoding proteins that are necessary for neurite formation or neuronal transmission, such as Actin beta, Tubulin alpha 1, Glutamate receptor, and Glutamine transporter. We further found a significant change in polysome association of rBYB1 in the brain of kainic acid treated mice and decrease of rB … More YB1 levels in KCl treated primary hippocampal neurons. These observations indicate that the Y-box protein regulates the translation of these mRNAs in neuronal activity dependent manner. In the future work, we will study the role(s) of rBYB1 in the neuronal activity dependent translational control. Furthermore, we examined the expression levels and polysome-bindings of the Y-box protein in various tissues, and observed different expression patterns in developing tissues. Identification of mRNAs will be the subject of further investigation.In cultured cell lines and/or tumor cells, a Y-box protein YB1 has been reported to modulate gene expression, including that of multidrug resistance gene (MDR1). YB1 recognizes an inverted CCAAT motif (Y-box sequence) on the promoter region of MDR1 gene. Because Y-box proteins can bind to both DNA and RNA, we investigated effects of nuclear RNA on DNA-binding of rBYB1 to the MDR1 promoter and found that nuclear RNA inhibits DNA-binding of rBYB1. Interestingly, nuclear RNA also inhibits DNA-binding of NF-Y, another protein known to be a transcription factor, which recognizes a Y-box sequence and participates in MDR1 expression. We used in vitro transcription system and found that nuclear RNA repressed the transcription from MDR1 promoter. These results suggest that nuclear RNA may take part in the regulation of MDR1 gene expression in neuroblastoma through affecting DNA-binding of the Y-box protein and NF-Y. RNA molecules should be isolated from brain or analised by SELEX. Less

为了研究Y-box蛋白RBYB1在细胞质和神经元细胞核中的功能，我们使用了大鼠脑提取物，原发性海马神经元和神经母细胞瘤NG108-15细胞的核。发现RBYB1在产后和早期产后大脑的细胞质中被认为是表达的，然后随着脑发育而发展为成人水平。 RBYB1在蔗糖密度梯度中的分布表明，该蛋白质通过RNA与多聚体相互作用。据报道，Y-box蛋白与mRNA结合并调节翻译。我们通过使用抗RBYB1抗体进行免疫沉淀与发育中的大脑中的RBYB1相互作用，并鉴定了一些编码神经蛋白形成或神经元传播所必需的mRNA，例如肌动蛋白β，小管蛋白alpha Alpha Alpha 1，谷氨酸受体和谷氨酰胺转运剂。我们进一步发现，RBYB1在Kainic Acid处理的小鼠大脑中的多元体相关性发生了显着变化，RB的降低……KCL处理过的原发性海马神经元中的Yb1水平越多。这些观察结果表明，Y-box蛋白在神经元活性依赖性方式中调节这些mRNA的翻译。在将来的工作中，我们将研究RBYB1在神经元活动依赖性翻译控制中的作用。此外，我们检查了各种组织中Y-box蛋白的表达水平和多膜结合，并观察到了发育中的组织中不同的表达模式。 MRNA的鉴定将是进一步研究的主题。在培养的细胞系和/或肿瘤细胞中，据报道，Y-box蛋白Yb1可以调节基因表达，包括多药抗性基因（MDR1）。 YB1识别MDR1基因启动子区域上的倒CCAAT基序（Y-box序列）。由于Y-box蛋白可以与DNA和RNA结合，因此我们研究了核RNA对RBYB1对MDR1启动子的DNA结合的影响，发现核RNA抑制RBYB1的DNA结合。有趣的是，核RNA还抑制了NF-Y的DNA结合，这是另一种已知是转录因子的蛋白质，它识别Y盒序列并参与MDR1表达。我们使用了体外转录系统，发现核RNA已从MDR1启动子中复制。这些结果表明，核RNA可以通过影响Y-box蛋白和NF-Y的DNA结合来调节神经母细胞瘤中MDR1基因的表达。 RNA分子应与大脑分离或通过SELEX分离。较少的