Templin Andrew T, Maier Bernhard, Tersey Sarah A, Hatanaka Masayuki, Mirmira Raghavendra G
Department of Cellular and Integrative Physiology (A.T.T., R.G.M.), Department of Pediatrics and the Herman B Wells Center for Pediatric Research (B.M., S.A.T., M.H., R.G.M.), Department of Biochemistry and Molecular Biology (R.G.M.), and Department of Medicine (R.G.M.), Indiana University School of Medicine, Indianapolis, Indiana 46202.
Mol Endocrinol. 2014 Nov;28(11):1820-30. doi: 10.1210/me.2014-1157. Epub 2014 Sep 24.
In type 1 diabetes, proinflammatory cytokines secreted by infiltrating immune cells activate the unfolded protein response (UPR) in islet β-cells, which leads to attenuation of global mRNA translation. Under such conditions, privileged mRNAs required for adaptation to the prevailing stress are maintained in an actively translated state. Pdx1 is a β-cell transcription factor that is required for the adaptive UPR, but it is not known how translation of its mRNA is maintained under these conditions. To study translation, we established conditions in vitro with MIN6 cells and mouse islets and a mixture of proinflammatory cytokines (IL-1β, TNF-α, and IFN-γ) that mimicked the UPR conditions seen in type 1 diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Similar to other privileged mRNAs (Atf4 and Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes under the UPR, whereas the mRNA encoding a proinsulin-processing enzyme (Cpe) and others partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5'-untranslated region of mouse Pdx1 (between bp -105 to -280) contained elements that promoted translation under both normal and UPR conditions, and this region exhibited conserved sequences and secondary structure similar to those of other known internal ribosome entry sites. Our findings suggest that Pdx1 protein levels are maintained in the setting of the UPR, in part, through elements in the 5'-untranslated region that confer privileged mRNA translation in a 5'-7-methylguanylate cap-independent manner.
在1型糖尿病中，浸润的免疫细胞分泌的促炎细胞因子激活胰岛β细胞中的未折叠蛋白反应（UPR），这导致整体mRNA翻译减弱。在这种情况下，适应当前应激所需的特殊mRNA保持在活跃翻译状态。Pdx1是一种β细胞转录因子，是适应性UPR所必需的，但尚不清楚其mRNA在这些条件下如何维持翻译。为了研究翻译，我们在体外建立了MIN6细胞、小鼠胰岛和促炎细胞因子混合物（IL-1β、TNF-α和IFN-γ）的条件，这些混合物模拟了1型糖尿病中所见的UPR条件。然后对细胞提取物进行多核糖体分析，以监测核糖体对mRNA占据情况的变化。与其他特殊mRNA（Atf4和Chop）类似，Pdx1 mRNA在UPR下仍分配在活跃翻译的多核糖体中，而编码胰岛素原加工酶（Cpe）的mRNA和其他mRNA则分配到非活跃翻译的单核糖体中。双顺反子荧光素酶报告基因分析表明，小鼠Pdx1的5'-非翻译区远端部分（-105至-280碱基对之间）包含在正常和UPR条件下均能促进翻译的元件，并且该区域呈现出与其他已知内部核糖体进入位点相似的保守序列和二级结构。我们的研究结果表明，在UPR环境中，Pdx1蛋白水平部分通过5'-非翻译区中的元件得以维持，这些元件以不依赖于5'-7-甲基鸟苷酸帽的方式赋予特殊mRNA翻译能力。
