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

在高等真核生物中，基因表达的各个步骤是相互影响的。例如，剪接不仅从前体mRNA中去除内含子，而且还在mRNA的外显子-外显子连接附近沉积外显子连接复合物EJC，从而增强下游事件，例如翻译、转运和无义介导的mRNA衰变（NMD）。Y14和Magoh是EJC的核心成分，它们与细胞核中的mRNA结合，并在细胞质中保持结合。这有力地表明这些蛋白质在RNA定位NMD中具有作用。事实上，果蝇中这些蛋白质的同源物已被证明参与卵母细胞中oskar mRNA的定位。本研究假设EJC也将mRNA剪接与mRNA在哺乳动物细胞中的定位联系起来，并一直在研究mRNA在神经元细胞中定位的分子机制。免疫组化染色证实，在神经炎的轴部有含有核帽结合复合物（CBC）和Y14的颗粒。这些颗粒与微管相连，并被认为是沿着微管沿着运输的。我们还发现了颗粒，含有细胞质帽结合蛋白，eIF 4 E，在神经元细胞的分支区域。通过进行免疫染色，我们证明了这两个颗粒含有Dcp 1a，这是P体的标志物。这些结果表明，在神经细胞中存在两种不同的p小体样颗粒，一种是含有可运输p小体的CBC-EJC，另一种是含有锚定p小体的eIF 4 E。我们的研究结果有力地表明了一种新的调节机制，本地翻译的本地mRNA在神经元中。

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.