Synonymous nucleotide modification of the KCNH2 gene affects both mRNA characteristics and translation of the encoded hERG ion channel
Synonymous nucleotide modification of the KCNH2 gene affects both mRNA characteristics and translation of the encoded hERG ion channel
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DOI：
10.1074/jbc.ra118.001805
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发表时间：
2018-08-03
影响因子：
4.8
通讯作者：
McDonald, Thomas, V
中科院分区：
文献类型：
--
作者：
Bertalovitz, Alexander C.;Badhey, Marika L. Osterbur;McDonald, Thomas, V
同义核苷酸变异越来越被认为是影响蛋白质表达的一个因素，但其潜在机制尚不完全清楚。在这里，我们研究了同义变化是否会影响钾电压门控通道亚家族 H 成员 2 (KCNH2) 基因的表达，该基因编码与遗传性心律失常相关的人 ether-a-go-go 相关基因 (hERG) 离子通道。我们检查了先前描述的合成版本（hERG-密码子修饰（CM）），其具有同义替换，旨在相对于天然构建体（hERG-NT）减少GC含量、稀有密码子和mRNA二级结构。在 HEK293T 细胞中，hERG-CM 的蛋白表达量低于 hERG-NT。我们发现 hERG-NT mRNA 的稳态丰度高于 hERG-CM，因为 hERG-NT 的转录率增强且 mRNA 稳定性增加。 hERG-CM 的翻译独立减少，有助于 hERG-NT 通道蛋白的总体合成量增加。然而，这被新合成的 hERG-NT 通道的更高聚集所部分抵消，导致产生无功能的蛋白质。 hERG-NT 和 hERG-CM 嵌合体的区域 mRNA 分析表明，编码区 5 个片段的同义变化在 mRNA 和蛋白质水平上对 hERG 合成影响最大。总而言之，这些结果表明编码区（特别是 hERG mRNA 的 5 区）内的同义核苷酸变异可以影响转录和翻译。这些发现支持这样的观点：在遗传性心脏病研究中分析 hERG DNA 序列时，应更多地关注同义遗传变异的影响。
Synonymous nucleotide variation is increasingly recognized as a factor than can affect protein expression, but the underlying mechanisms are incompletely understood. Here, we investigated whether synonymous changes could affect expression of the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene, encoding the human ether-a-go-go-related gene (hERG) ion channel, which is linked to hereditary cardiac arrhythmia. We examined a previously described synthetic version (hERG-codon modified (CM)) with synonymous substitutions designed to reduce GC content, rare codons, and mRNA secondary structure relative to the native construct (hERG-NT). hERG-CM exhibited lower protein expression than hERG-NT in HEK293T cells. We found that the steady-state abundance of hERG-NT mRNA was greater than hERG-CM because of an enhanced transcription rate and increased mRNA stability for hERG-NT. Translation of hERG-CM was independently reduced, contributing to the overall greater synthesis of hERG-NT channel protein. This was partially offset, however, by a higher aggregation of a newly synthesized hERG-NT channel, resulting in nonfunctional protein. Regional mRNA analyses of chimeras of hERG-NT and hERG-CM revealed that synonymous changes in the 5 segments of the coding region had the greatest influence on hERG synthesis at both the mRNA and protein levels. Taken together, these results indicate that synonymous nucleotide variations within the coding region, particularly in the 5 region of the hERG mRNA, can affect both transcription and translation. These findings support the notion that greater attention should be given to the effects of synonymous genetic variation when analyzing hERG DNA sequences in the study of hereditary cardiac disease.