Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy.
Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy.
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DOI：
10.1261/rna.055749.115
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发表时间：
2016-07
期刊：
影响因子：
--
通讯作者：
Shanley DP
中科院分区：
文献类型：
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作者：
Zupanic A;Meplan C;Huguenin GV;Hesketh JE;Shanley DP
硒蛋白，一组特定的蛋白质，纳入硒代半胱氨酸的表达，分级调节硒的可用性，与一些，但不是所有的硒蛋白mRNA转录减少的丰度减少硒。硒代半胱氨酸插入到肽链中发生在翻译过程中，随后重新编码内部UGA终止密码子。越来越多的证据表明，这种UGA重编码与过早的翻译终止竞争，随后是转录本的无义介导的衰变（NMD）。在这项研究中，我们测试的假设，不同的硒蛋白mRNA的敏感性提前终止在翻译和差异敏感性硒蛋白转录NMD的硒蛋白层次结构的主要因素。硒蛋白转录丰度测定在Caco-2细胞中使用实时PCR在不同的硒条件下，得到的数据拟合的数学模型硒蛋白翻译。包括硒蛋白转录物对NMD的不同敏感性和非NMD相关的过早翻译终止的不同频率的组合的校准模型能够拟合所有测量。使用SiRNA敲低关键NMD因子UPF 1（上移码蛋白1）和硒蛋白mRNA表达的表达来测试模型预测。校准模型能够预测UPF 1敲除对所有测试的硒蛋白基因表达的影响，除了SPS 2（硒磷酸合成酶），这本身是必不可少的硒蛋白合成。这些结果表明NMD在硒蛋白mRNA的分级调控中具有重要作用，但SPS 2除外，其表达可能受不同机制的调控。
The expression of selenoproteins, a specific group of proteins that incorporates selenocysteine, is hierarchically regulated by the availability of Se, with some, but not all selenoprotein mRNA transcripts decreasing in abundance with decreasing Se. Selenocysteine insertion into the peptide chain occurs during translation following recoding of an internal UGA stop codon. There is increasing evidence that this UGA recoding competes with premature translation termination, which is followed by nonsense-mediated decay (NMD) of the transcript. In this study, we tested the hypothesis that the susceptibility of different selenoprotein mRNAs to premature termination during translation and differential sensitivity of selenoprotein transcripts to NMD are major factors in the selenoprotein hierarchy. Selenoprotein transcript abundance was measured in Caco-2 cells using real-time PCR under different Se conditions and the data obtained fitted to mathematical models of selenoprotein translation. A calibrated model that included a combination of differential sensitivity of selenoprotein transcripts to NMD and different frequency of non-NMD related premature translation termination was able to fit all the measurements. The model predictions were tested using SiRNA to knock down expression of the crucial NMD factor UPF1 (up-frameshift protein 1) and selenoprotein mRNA expression. The calibrated model was able to predict the effect of UPF1 knockdown on gene expression for all tested selenoproteins, except SPS2 (selenophosphate synthetase), which itself is essential for selenoprotein synthesis. These results indicate an important role for NMD in the hierarchical regulation of selenoprotein mRNAs, with the exception of SPS2 whose expression is likely regulated by a different mechanism.