Bukowy-Bieryllo Zuzanna, Dabrowski Maciej, Witt Michał, Zietkiewicz Ewa
a Institute of Human Genetics, Polish Academy of Sciences , Poznan , Poland.
RNA Biol. 2016 Oct 2;13(10):1041-1050. doi: 10.1080/15476286.2016.1219832. Epub 2016 Aug 12.
Translational readthrough of premature termination codons (PTCs) induced by pharmacological compounds has proven to be an effective way of restoring functional protein expression and reducing symptoms in several genetic disorders. We tested the potential of different concentrations of several aminoglycosides (AAGs) for promoting PTC-readthrough in 5 genes involved in the pathogenesis of primary ciliary dyskinesia, an inherited disorder caused by the dysfunction of motile cilia and flagella. The efficiency of readthrough stimulation of PTCs cloned in dual reporter vectors was examined in 2 experimental settings: in vitro (transcription/translation system) and ex vivo (transiently transfected epithelial cell line). PTC-readthrough was observed in 5 of the 16 mutations analyzed. UGA codons were more susceptible to AAG-stimulated readthrough than UAG; no suppression of UAA was observed. The efficiency of PTC-readthrough in vitro (from less than 1% to ∼28% of the translation from the corresponding wild-type constructs) differed with the AAG type and concentration, and depended on the combination of AAG and PTC, indicating that each PTC has to be individually tested with a range of stimulating compounds. The maximal values of PTC suppression observed in the ex vivo experiments were, depending on AAG used, 3-5 times lower than the corresponding values in vitro, despite using AAG concentrations that were 2 orders of magnitude higher. This indicates that, while the in vitro system is sufficient to examine the readthrough-susceptibility of PTCs, it is not sufficient to test the compounds potential to stimulate PTC-readthrough in the living cells. Most of the tested compounds (except for G418) at their highest concentrations did not disturb ciliogenesis in the cultures of primary respiratory epithelial cells from healthy donors.
药理学化合物诱导的提前终止密码子（PTC）的翻译通读已被证明是恢复功能性蛋白质表达并减轻几种遗传疾病症状的有效方法。我们测试了几种不同浓度的氨基糖苷类药物（AAG）促进5个与原发性纤毛运动障碍发病机制相关基因中PTC通读的潜力，原发性纤毛运动障碍是一种由活动纤毛和鞭毛功能障碍引起的遗传性疾病。在两种实验环境中检测了克隆到双报告载体中的PTC通读刺激效率：体外（转录/翻译系统）和离体（瞬时转染的上皮细胞系）。在所分析的16个突变中有5个观察到了PTC通读。UGA密码子比UAG密码子更容易受到AAG刺激的通读影响；未观察到UAA的抑制。体外PTC通读效率（从相应野生型构建体翻译的不到1%至约28%）因AAG类型和浓度而异，并取决于AAG和PTC的组合，这表明每个PTC都必须用一系列刺激化合物单独测试。尽管使用的AAG浓度高2个数量级，但离体实验中观察到的PTC抑制最大值，取决于所使用的AAG，比体外相应值低3至5倍。这表明，虽然体外系统足以检测PTC的通读敏感性，但不足以测试化合物在活细胞中刺激PTC通读的潜力。大多数测试化合物（除G418外）在其最高浓度下不会干扰健康供体的原代呼吸道上皮细胞培养物中的纤毛发生。
