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

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.

Figure 1

Transfection efficiency of 293 cells. (A) Cells under the light microscope. (B) Expression in the nucleus using DAPI. (C) Cells successfully transfected with GFAP and wild-type plasmids. Scale bar, 100 µm.

Figure 2

R1014X and W927X mutants. (A) Nucleotide sequence surrounding the stop codon. (B) R1014X and W927X mutants containing different stop codons (TGA, TAA and TAG within boxes) and different +4 nucleotides immediately downstream of the stop codon, TGA, are shown. The single-letter abbreviations of the amino acids are as follows: A, alanine; R, arginine; C, cysteine; Q, glutamine; E, glutamic acid; G, glycine; L, leucine; P, proline; S, serine; T, threonine; W, tryptophan; Y, tyrosine.

Figure 3

Protein expression of HERG in the control and HERG-WT groups before and after G418 treatment in 293 cells. Control group (transfection reagent only): (A) no antibody added before G418 treatment; (B) antibody against the N-terminus of HERG protein added before G418; (C) anti-C-terminal antibody added before G418 treatment; (D) anti-C-terminal antibody added following treatment with G418. HERGWT group: (E) no antibody added before G418; (F) antibody against the N-terminus of HERG protein added before G418; (G) anti-C-terminal antibody added before G418 treatment; (H) anti-C-terminal antibody following treatment with G418. Red fluorescence represents the expression of HERG protein. Scale bar, 100 µm. HERG, human ether-a-go-go-related; WT, wild-type.

Figure 4

Protein expression of the HERG-R1014X mutant with stop codon TGA, TAA or TAG prior to and following G418 treatment in 293 cells. Antibody raised against the N-terminus of the HERG protein was used before G418 was added in the (A) TGA, (B) TAA and (C) TAG groups. Antibody against the C-terminus of the HERG protein was used before G418 was added to the (D) TGA, (E) TAA and (F) TAG groups. Following treatment with G418, anti-C-erminal antibody was used in the (G) TGA, (H) TAA and (I) TAG groups. Red fluorescence represents the expression of HERG protein. Scale bar, 100 µm. HERG, human ether-a-go-go-related.

Figure 5

Ionic currents recorded for the WT and R1014X mutant HERG channels before and after G418 treatment in transfected 293 cells. (A) Representative families of current traces recorded from cells transfected with WT or the three mutant HERG cDNAs are shown. (B) Peak tail currents recorded for the cells transfected with WT or R1014X mutant proteins with different stop codons. ^*P<0.05. HERG, human ether-a-go-go-related; WT, wild-type.

Figure 6

Protein expression levels in cells transfected with HERG-W927X mutant with stop codon TGA, TAA, or TAG, respectively, before and after G418 treatment. (A) TGA, (B) TAA and (C) TAG groups in which antibody raised against the N-terminus of the HERG protein was used before G418. (D) TGA, (E) TAA and (F) TAG groups in which antibody against the C-terminus of the HERG protein was used before G418. (G) TGA, (H) TAA and (I) TAG groups in which anti-C-terminal antibody was used following treatment with G418. Red fluorescence represents the expression of the HERG protein. Scale bar, 100 µm. HERG, human ether-a-go-go-related.

Figure 7

Ionic current recorded from W927X mutant HERG channels before and after G418 treatment in transfected 293 cells. (A) Representative families of current traces recorded from cells transfected with WT or the three mutant HERG cDNAs. (B) Peak tail currents from cells transfected with the W927X mutants featuring the different stop codons. ^*P<0.05. HERG, human ether-a-go-go-related; WT, wild-type.

Figure 8

Protein expression in cells transfected with HERG-R1014X mutant containing different +4 nucleotides downstream of the stop codon TGA before and after G418 treatment. Antibody against the N-terminus of the protein was used before G418 was added to mutants with the +4 nucleotides (A) T, (B) G, (C) A and (D) C, respectively. Antibody against the C-terminus of the protein was used before mutants with the +4 nucleotides (E) T, (F) G, (G) A and (H) C were treated with G418. (I-L) Following treatment with G418, anti-C-terminal antibody was used for the (I) T, (J) G, (K) A and (L) C mutants. Red fluorescence represents expression of the HERG protein. Scale bar, 100 µm. HERG, human ether-a-go-go-related.

Figure 9

Ionic current recorded from the R1014X mutant HERG channels before and after G418 treatment of the transfected 293 cells. (A) Representative families of current traces recorded from cells transfected with the four mutant HERG cDNAs. (B) Peak tail currents from cells transfected with the R1014X mutants with the different +4 nucleotides downstream of the stop codon TGA. ^*P<0.05. HERG, human ether-a-go-go-related; WT, wild-type.