Linde Liat, Kerem Batsheva
Department of Genetics, The Life Sciences Institute, The Hebrew University, 91904, Jerusalem, Israel.
Methods Mol Biol. 2011;741:137-54. doi: 10.1007/978-1-61779-117-8_10.
Approximately one-third of the alleles causing genetic diseases carry premature termination codons (PTCs). Therapeutic approaches for mutations generating in-frame PTCs are aimed at promoting translational readthrough of the PTC, to enable the synthesis and expression of full-length functional proteins. Interestingly, readthrough studies in tissue culture cells, mouse models, and clinical trials revealed a wide variability in the response to the readthrough treatments. The molecular basis for this variability includes the identity of the PTC and its sequence context, the chemical composition of the readthrough drug, and, as we showed recently, the level of PTC-bearing transcripts. One post-transcriptional mechanism that specifically regulates the level of PTC-bearing transcripts is nonsense-mediated mRNA decay (NMD). We have previously shown a role for NMD in regulating the response of CF patients carrying CFTR PTCs to readthrough treatment. Here we describe all the protocols for analyzing CFTR nonsense transcript levels and for investigating the role of NMD in the response to readthrough treatment. This includes inhibition of the NMD mechanism, quantification of CFTR nonsense transcripts and physiologic NMD substrates, and analysis of the CFTR function.
约三分之一导致遗传疾病的等位基因携带过早终止密码子（PTC）。针对产生框内PTC的突变的治疗方法旨在促进PTC的翻译通读，以实现全长功能蛋白的合成和表达。有趣的是，在组织培养细胞、小鼠模型和临床试验中的通读研究表明，对通读治疗的反应存在很大差异。这种差异的分子基础包括PTC的身份及其序列背景、通读药物的化学成分，以及正如我们最近所表明的，携带PTC的转录本水平。一种特异性调节携带PTC的转录本水平的转录后机制是非义介导的mRNA降解（NMD）。我们之前已经证明NMD在调节携带CFTR PTC的CF患者对通读治疗的反应中起作用。在这里，我们描述了所有用于分析CFTR无义转录本水平以及研究NMD在通读治疗反应中的作用的方案。这包括抑制NMD机制、定量CFTR无义转录本和生理性NMD底物，以及分析CFTR功能。
