Abstract
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.
Highlights
Synonymous nucleotide variation is increasingly recognized as a factor than can affect protein expression, but the underlying mechanisms are incompletely understood
We sought to investigate whether synonymous modification of KCNH2 could impact human ether-a-go-go–related gene (hERG) protein expression
We had previously demonstrated that hERG-CM had reduced protein expression relative to hERG construct (hERG-NT) and that hERG-CM trafficked more efficiently to the cell surface than hERG-NT
Summary
HERG-CM protein generated from new vector backbone consistently yields decreased protein expression compared with hERG-NT. The half-life of hERG-NT mRNA was ϳ2-fold greater than that of hERG-CM (4.0 versus 2.1 h, respectively) (Fig. 2C) ( p Ͻ 0.0001) This result indicates that synonymous modifications can affect mRNA stability. The “first” position is comparing the average protein expression of NNC, NCN, and NCC (chimeras containing the first third of NT) with CNC, CNN, and CCN (chimeras containing the first third of CM) In this analysis, the 5Ј third of the mRNA appeared to have the greatest influence on protein translation (Fig. 8, B and C) because the presence of the hERG-NT sequence in the 5Ј region yielded higher protein expression regardless of the composition of the remaining twothirds of the protein. This provides evidence to the hypothesis that synonymous modification in localized regions of the cDNA of hERG can influence synthesis both at the mRNA and protein levels and that the 5Ј-coding region, the first 51 nucleotides, of hERG is essential in maintaining native mRNA and protein expression profiles
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