Stop codons and the +4 nucleotide may influence the efficiency of G418 in rescuing nonsense mutations of the HERG gene

Yu,Haiyun; Meng,Yanhai; Zhang,Shuhong; Tian,Chen; Wu,Fang; Li,Ning; Li,Qiuyang; Jin,Yulan; Pu,Jielin

doi:10.3892/ijmm.2019.4360

Stop codons and the +4 nucleotide may influence the efficiency of G418 in rescuing nonsense mutations of the HERG gene

Authors:
- Haiyun Yu
- Yanhai Meng
- Shuhong Zhang
- Chen Tian
- Fang Wu
- Ning Li
- Qiuyang Li
- Yulan Jin
- Jielin Pu
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Affiliations: Department of Pathology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, P.R. China, Department of ICU, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China, Department of Pathology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, P.R. China, Department of Pathology, Capital Medical University Electric Power Teaching Hospital (State Grid Corporation Beijing Electric Power Hospital), Beijing 100037, P.R. China, Department of Cardiology, East Hospital, Tongji University, Shanghai 200120, P.R. China
Published online on: October 1, 2019 https://doi.org/10.3892/ijmm.2019.4360
Pages: 2037-2046
Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The importance of the local sequence context in determining how efficiently aminoglycosides rescue nonsense mutations has been established previously in disease models. Different stop codons appear to facilitate the termination process with differing efficiencies. Furthermore, the efficiency with which termination is suppressed may also be influenced by the local sequence context surrounding the stop codon. The strongest bias has usually been identified with the nucleotide base that immediately follows the stop codon in the majority of experiments. However, how the sequence context influences the efficiency of aminoglycosides in rescuing the human ether‑a‑go‑go‑related (HERG) protein in mammalian cells remains to be fully elucidated. Therefore, the present study was devised to examine the susceptibility of different termination codons on the HERG gene and the +4 nucleotide immediately following them to be suppressed by aminoglycosides in 293 cells. The 293 cells were transiently transfected with the wild‑type or mutant genes. The read‑through effect was subsequently examined by adding aminoglycoside G418 into the culture medium, followed by incubation of the cells for 24 h. An immunofluorescence method was then used to observe the protein expression of HERG prior to and following drug treatment. Patch clamping was performed to evaluate the function of the HERG protein. These experiments revealed that stop codons TGA and TAA in the R1014X mutant were more susceptible to treatment with the drug G418. Similar results were observed with the W927X‑TGA and W927X‑TAA mutants. Subsequently, R1014X‑TGAC, R1014X‑TGAG and R1014X‑TGAA mutants were constructed based on the R1014X‑TGAT mutant. The level of red fluorescence was observed prior to and following the administration of G418 using antibodies targeting the N‑ or C‑terminus of the HERG protein. However, the tail current density was found only to increase with the R1014X‑TGAT mutant following G418 treatment. Taken together, the results of the present study suggest that the type of premature stop codon and the context of the nucleotide immediately following at the +4 position, may determine the pharmacological rescue efficiency of the HERG gene.

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