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Abstract：
Rett syndrome (RTT) is a neurodevelopmental disorder resulting from genetic mutations in the methyl CpG binding protein 2 (MeCP2) gene. Specifically, around 35% of RTT patients harbor premature termination codons (PTCs) within the MeCP2 gene due to nonsense mutations. A promising therapeutic avenue for these individuals involves the use of aminoglycosides, which stimulate translational readthrough (TR) by causing stop codons to be interpreted as sense codons. However, the effectiveness of this treatment depends on several factors, including the type of stop codon and the surrounding nucleotides, collectively referred to as the stop codon context (SCC). Here, we develop a high-content reporter system to precisely measure TR efficiency at different SCCs, assess the recovery of the full-length MeCP2 protein, and evaluate its subcellular localization. We have conducted a comprehensive investigation into the intricate relationship between SCC characteristics and TR induction, examining a total of 14 pathogenic MeCP2 nonsense mutations with the aim to advance the prospects of personalized therapy for individuals with RTT. Our results demonstrate that TR induction can successfully restore full-length MeCP2 protein, albeit to varying degrees, contingent upon the SCC and the specific position of the PTC within the MeCP2 mRNA. TR induction can lead to the re-establishment of nuclear localization of MeCP2, indicating the potential restoration of protein functionality. In summary, our findings underscore the significance of SCC-specific approaches in the development of tailored therapies for RTT. By unraveling the relationship between SCC and TR therapy, we pave the way for personalized, individualized treatment strategies that hold promise for improving the lives of individuals affected by this debilitating neurodevelopmental disorder. KEY MESSAGES: The efficiency of readthrough induction at MeCP2 premature termination codons strongly depends on the stop codon context. The position of the premature termination codon on the transcript influences the readthrough inducibility. A new high-content dual reporter assay facilitates the measurement and prediction of readthrough efficiency of specific nucleotide stop contexts. Readthrough induction results in the recovery of full-length MeCP2 and its re-localization to the nucleus. MeCP2 requires only one of its annotated nuclear localization signals.
摘要：
Rett综合征（RTT）是一种神经发育障碍，由甲基CpG结合蛋白2（MeCP2）基因的基因突变引起。具体来说,由于无义突变,约35%的RTT患者在MeCP2基因内携带过早终止密码子(PTC).对这些人来说，一个有希望的治疗途径包括使用氨基糖苷类，通过使终止密码子被解释为有义密码子来刺激翻译连读（TR）。然而,这种治疗的有效性取决于几个因素，包括终止密码子的类型和周围的核苷酸，统称为终止密码子上下文(SCC)。这里,我们开发了一个高内容报告系统来精确测量不同SCC的TR效率，评估全长MeCP2蛋白的回收率，并评估其亚细胞定位。我们对SCC特征与TR诱导之间的复杂关系进行了全面的研究，检查总共14个致病性MeCP2无义突变，目的是提高RTT患者个性化治疗的前景。我们的结果表明，TR诱导可以成功恢复全长MeCP2蛋白，尽管程度不同，取决于SCC和PTC在MeCP2mRNA中的特定位置。TR诱导可导致MeCP2核定位的重建,表明蛋白质功能性的潜在恢复。总之,我们的发现强调了SCC特异性治疗方法在开发RTT定制治疗中的重要性.通过解开SCC和TR治疗之间的关系，我们为个性化铺平道路，个性化治疗策略有望改善受这种使人衰弱的神经发育障碍影响的个体的生活。关键信息：MeCP2提前终止密码子的连读诱导效率强烈依赖于终止密码子的上下文。转录物上提前终止密码子的位置影响连读诱导性。一种新的高含量双报告子测定法有助于测量和预测特定核苷酸终止上下文的通读效率。读出诱导导致全长MeCP2的恢复及其重新定位到细胞核。MeCP2仅需要其注释的核定位信号之一。
