Abstract

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGR^ex1-19 and RPGR^ORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGR^ORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.

中文翻译：

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X连锁色素性视网膜炎 (XLRP) 中 RPGR 亚型不平衡导致外显子 ORF15 突变导致纤毛缺陷

 抽象的

色素性视网膜炎 GTP 酶调节因子 ( RPGR ) 突变会导致严重的视网膜纤毛病，即 X 连锁色素性视网膜炎。尽管表达了两种主要的选择性剪接异构体 RPGR ^ex1-19和 RPGR ^ORF15 ，但这些异构体在疾病发病机制中的相对重要性尚不清楚。在这里，我们分析了八名患者的成纤维细胞样本，发现他们都比正常对照形成了更长的纤毛，尽管程度不同。尽管所有突变体RPGRORF15信使 RNA (mRNA) 都不稳定，但它们的稳态水平与对照细胞中的水平相似或更高，表明转录可能增加。三个具有较高水平突变RPGRORF15 mRNA 的成纤维细胞也表现出显着较高水平的RPGRex1-19 mRNA。四个RPGRex1-19水平未改变的样本携带RPGRORF15突变，导致该亚型相对不稳定。因此，在所有情况下， RPGRex1-19 / RPGRORF15同工型比率均增加，这与纤毛延伸缺陷高度相关。此外， RPGRex1-19 （模拟RPGRex1-19与RPGRORF15亚型比例的增加）或RPGRORF15 （模拟该比例的减少）的过度表达分别导致纤毛显着更长或更短。 值得注意的是，纤毛长度缺陷似乎可归因于野生型 RPGR ^ORF15蛋白的丢失和RPGRex1-19亚型的较高水平，表明观察到的缺陷是由于亚型比率改变所致。这些结果表明，维持最佳的RPGRex1-9与RPGRORF15比率对于纤毛生长至关重要，并且设计专注于恢复RPGRex1-19 / RPGRORF15比率的最佳方法的策略可能会带来更好的治疗结果。