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Analysis of mRNA localization mechanism in neuronal cells mediated by splicing-dependent complex, EJC

剪接依赖性复合物EJC介导的神经元细胞mRNA定位机制分析

基本信息

批准号：
16370078
负责人：
KATAOKA Naoyuki
金额：
$ 9.03万
依托单位：
Tokyo Medical and Dental University (2007)Kyoto University (2004-2006)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

项目摘要

It has been well accepted that steps of gene expression influence to each other in higher eukaryotes. For example, splicing not only removes intron from pre-mRNAs but also deposits exon junction complex near exon-exon junction of mRNA EJC enhances downstream events, such as translation, transport and nonsense mediated mRNA decay (NMD). Y14 and Magoh, core components of EJC, bind to mRNA in the nucleus and remain bound in the cytoplasm. This has strongly suggested that these proteins have roles in RNA localization NMD. Indeed, homologs of these proteins in Drosophila have been shown to be involved in oskar mRNA localization in oocytes. In addition, others and we also showed that these proteins participate in NMD.In this research, we hypothesized that EJC also links mRNA splicing with mRNA localization in mammalian cells, and have been investigating the molecular mechanism of mRNA localization in neuronal cells. We have identified by immunostaining that there are granules that contain nuclear cap binding complex (CBC) and Y14 in the shaft regions of neuritis. These granules are associated with microtubules and thought to be transported along them. We also found granules that contain cytoplasmic cap binding protein, eIF4E, in the branched regions of neuronal cells. By performing immunostaining, we demonstrated that both granules contain Dcp1a, which is a marker of P body. These results indicate that there are two different p body-like granules in neuronal cells; one is CBC-EJC containing transportable p body, the other is eIF4E containing anchored p body. Our findings strongly suggest a new regulatory mechanism of local translation for localized mRNAs in neurons.

在高等真核生物中，基因表达的步骤是相互影响的，这一点已经被广泛接受。例如，剪接不仅从pre-mRNA中去除内含子，而且在mRNA EJC的外显子-外显子连接处沉积外显子连接复合体，增强了下游事件，如翻译、转运和无义介导的mRNA衰变（NMD）。Y14和Magoh是EJC的核心成分，在细胞核中与mRNA结合，并在细胞质中保持结合。这强烈提示这些蛋白在RNA定位NMD中起作用。事实上，这些蛋白在果蝇体内的同源物已被证明与oskar mRNA在卵母细胞中的定位有关。此外，我们和其他人也发现这些蛋白参与了NMD。在本研究中，我们假设EJC在哺乳动物细胞中也将mRNA剪接与mRNA定位联系起来，并对神经元细胞中mRNA定位的分子机制进行了研究。我们通过免疫染色发现，在神经炎的轴区存在含有核帽结合复合物（CBC）和Y14的颗粒。这些颗粒与微管有关，并被认为是沿着微管运输的。我们还在神经细胞的分支区发现了含有细胞质帽结合蛋白eIF4E的颗粒。通过免疫染色，我们发现两种颗粒都含有Dcp1a，这是P体的标记物。这些结果表明，神经元细胞中存在两种不同的p体样颗粒；一种是含有可移动p体的CBC-EJC，另一种是含有锚定p体的eIF4E。我们的发现有力地提示了神经元中局部mrna翻译的一种新的调控机制。