Roman-Sanchez Ramon, Wensel Theodore G, Wilson John H
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, USA.
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, USA.
Exp Eye Res. 2016 Apr;145:444-449. doi: 10.1016/j.exer.2015.09.013. Epub 2015 Sep 26.
Eight different nonsense mutations in the human rhodopsin gene cause retinitis pigmentosa (RP), an inherited degenerative disease of the retina that can lead to complete blindness. Although all these nonsense mutations lead to premature termination codons (PTCs) in rhodopsin mRNA, some display dominant inheritance, while others are recessive. Because nonsense-mediated decay (NMD) can degrade mRNAs containing PTCs and modulate the inheritance patterns of genetic diseases, we asked whether any of the nonsense mutations in the rhodopsin gene generated mRNAs that were susceptible to degradation by NMD. We hypothesized that nonsense mutations that caused mild RP phenotypes would trigger NMD, whereas those that did not engage NMD would cause more severe RP phenotypes-presumably due to the toxicity of the truncated protein. To test our hypothesis, we transfected human rhodopsin nonsense mutants into HEK293 and HT1080 human cells and measured transcript levels by qRT-PCR. In both cell lines, rhodopsin mutations Q64X and Q344X, which cause severe phenotypes that are dominantly inherited, yielded the same levels of rhodopsin mRNA as wild type. By contrast, rhodopsin mutations W161X and E249X, which cause recessive RP, showed decreased rhodopsin mRNA levels, consistent with NMD. Rhodopsin mutant Y136X, a dominant mutation that causes late-onset RP with a very mild pathology, also gave lower mRNA levels. Treatment of cells with Wortmannin, an inhibitor of NMD, eliminated the degradation of Y136X, W161X, and E249X rhodopsin mRNAs. These results suggest that NMD modulates the severity of RP in patients with nonsense mutations in the rhodopsin gene.
人类视紫红质基因中的八种不同无义突变会导致色素性视网膜炎（RP），这是一种遗传性视网膜退行性疾病，可导致完全失明。尽管所有这些无义突变都会导致视紫红质mRNA出现过早终止密码子（PTC），但有些表现为显性遗传，而另一些则为隐性遗传。由于无义介导的衰变（NMD）可以降解含有PTC的mRNA并调节遗传疾病的遗传模式，我们询问视紫红质基因中的任何无义突变是否会产生易被NMD降解的mRNA。我们假设导致轻度RP表型的无义突变会触发NMD，而那些不参与NMD的突变会导致更严重的RP表型，这可能是由于截短蛋白的毒性所致。为了验证我们的假设，我们将人类视紫红质无义突变体转染到HEK293和HT1080人类细胞中，并通过qRT-PCR测量转录水平。在这两种细胞系中，导致严重显性遗传表型的视紫红质突变Q64X和Q344X产生的视紫红质mRNA水平与野生型相同。相比之下，导致隐性RP的视紫红质突变W161X和E249X显示视紫红质mRNA水平降低，这与NMD一致。视紫红质突变体Y136X是一种导致迟发性RP且病理非常轻微的显性突变，其mRNA水平也较低。用NMD抑制剂渥曼青霉素处理细胞消除了Y136X、W161X和E249X视紫红质mRNA的降解。这些结果表明，NMD调节视紫红质基因无义突变患者中RP的严重程度。
