Pedder Christopher M, Ford Dianne, Hesketh John E
Institute for Cell and Molecular Biosciences, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Biochem Soc Trans. 2008 Jun;36(Pt 3):525-7. doi: 10.1042/BST0360525.
mRNA stability, mRNA translation and spatial localization of mRNA species within a cell can be governed by signals in the 3'-UTR (3'-untranslated region). Local translation of proteins is essential for the development of many eukaryotic cell types, such as the Drosophila embryo, where the spatial and temporal localization of bicoid and gurken mRNAs, among others, is required to establish morphogen gradients. More recent studies have suggested that mRNA localization also occurs with transcripts coding for membrane-based or secreted proteins, and that localization at organelles such as the endoplasmic reticulum directs translation more efficiently to specific subdomains, so as to aid correct protein localization. In human epithelial cells, the mRNA coding for SGLT1 (sodium-glucose co-transporter 1), an apical membrane protein, has been shown to be localized apically in polarized cells. However, the nature of the signals and RNA-binding proteins involved are unknown. Ongoing work is aimed at identifying the localization signals in the SGLT1 3'-UTR and the corresponding binding proteins. Using a protein extract from polarized Caco-2 cells, both EMSAs (electrophoretic mobility-shift assays) and UV cross-linking assays have shown that a specific protein complex is formed with the first 300 bases of the 3'-UTR sequence. MFold predictions suggest that this region folds into a complex structure and ongoing studies using a series of strategic deletions are being carried out to identify the precise nature of the motif involved, particularly the role of the sequence or RNA secondary structure, as well as to identify the main proteins present within the complex. Such information will provide details of the post-transcriptional events that lead to apical localization of the SGLT1 transcript and may reveal mechanisms of more fundamental importance in the apical localization of proteins in polarized epithelia.
mRNA的稳定性、mRNA翻译以及细胞内mRNA种类的空间定位可由3'-UTR（3'-非翻译区）中的信号调控。蛋白质的局部翻译对于许多真核细胞类型的发育至关重要，比如果蝇胚胎，其中，双胸蛋白和gurken mRNA等的时空定位对于建立形态发生素梯度是必需的。最近的研究表明，mRNA定位也发生在编码膜结合蛋白或分泌蛋白的转录本中，并且在内质网等细胞器处的定位能更有效地将翻译导向特定亚结构域，从而有助于蛋白质的正确定位。在人上皮细胞中，编码顶端膜蛋白SGLT1（钠-葡萄糖共转运蛋白1）的mRNA已被证明在极化细胞中定位于顶端。然而，所涉及的信号和RNA结合蛋白的性质尚不清楚。正在进行的工作旨在确定SGLT1 3'-UTR中的定位信号以及相应的结合蛋白。使用来自极化的Caco-2细胞的蛋白质提取物，电泳迁移率变动分析（EMSA）和紫外线交联分析均表明，一种特定的蛋白质复合物与3'-UTR序列的前300个碱基形成。MFold预测表明该区域折叠成复杂结构，并且正在使用一系列策略性缺失进行研究，以确定所涉及基序的确切性质，特别是序列或RNA二级结构的作用，以及确定复合物中存在的主要蛋白质。这些信息将提供导致SGLT1转录本顶端定位的转录后事件的细节，并可能揭示在极化上皮细胞中蛋白质顶端定位方面更具根本重要性的机制。
