Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN, USA.
RNA Biol. 2013 Feb;10(2):256-66. doi: 10.4161/rna.23022. Epub 2013 Jan 25.
The opioid receptors are among the most highly studied members of the superfamily of G-protein coupled receptors. Morphine and endogenous mu opioid peptides exert their pharmacological actions mainly through the mu opioid receptor (MOR). Expression of opioid receptor proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. Here, we demonstrate for the first time the role of vimentin as a post-transcriptional repressor in MOR gene regulation. To identify potential regulators of the mouse MOR gene, we performed affinity column chromatography using 5'-UTR-specific RNA oligonucleotides using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified an intermediate filament protein, vimentin, which bound specifically to the region between -175 and -150 (175-150) of the MOR 5'-UTR. Binding was confirmed by western blot analysis and RNA supershift assay. Furthermore, a cotransfection study demonstrated that the presence of vimentin resulted in reduced expression of the mouse MOR. Our data suggest that vimentin functions as a repressor of MOR translation, dependent on 175-150 of the MOR 5'-UTR.
阿片受体是 G 蛋白偶联受体超家族中研究最多的成员之一。吗啡和内源性 μ 阿片肽主要通过 μ 阿片受体 (MOR) 发挥其药理学作用。阿片受体蛋白的表达受广泛的转录和转录后加工调控。先前发现，小鼠 MOR 的 5'-非翻译区 (UTR) 对于神经元细胞中 MOR 基因的转录后调控很重要。在这里，我们首次证明了波形蛋白作为 MOR 基因调节中的转录后抑制剂的作用。为了鉴定小鼠 MOR 基因的潜在调节剂，我们使用神经母细胞瘤 NS20Y 细胞使用 5'-UTR 特异性 RNA 寡核苷酸进行了亲和柱层析。层析后进行二维凝胶电泳和 MALDI-TOF 质谱分析。我们鉴定出一种中间丝蛋白波形蛋白，它特异性结合 MOR 5'-UTR 的-175 到-150 （175-150）区域。通过 Western blot 分析和 RNA 超迁移实验证实了结合。此外，共转染研究表明，波形蛋白的存在导致小鼠 MOR 的表达减少。我们的数据表明，波形蛋白作为 MOR 翻译的抑制剂发挥作用，依赖于 MOR 5'-UTR 的 175-150 。
