Abstract 17243: Synonymous Modification of hERG Regulates Translation Rate and RNA Stability

Introduction/HypothesisSuppression (genetic or pharmacologic) of hERG channel expression diminishes the rapid delayed rectifying potassium current elevating risk for ventricular arrhythmia. Synonymous DNA variation (that does not alter amino acid coding) is increasingly recognized to influence gene expression. We codon-modified the hERG cDNA (hERG-CM) sequence to reduce the GC-content, rare codon usage, and mRNA secondary structure. Our previous studies showed that this synonymous codon modification influenced all aspects of channel synthesis (transcription, translation, mRNA stability, protein folding, and trafficking). Here we sought to further elucidate the mechanism of synonymous nucleotide variation on translation of the hERG channel protein.Methods/ResultsAt steady-state, hERG-CM was produced ~2-fold less than native hERG (hERG-NT) when cRNA was directly transfected into HEK293 cells (thereby eliminating effects on transcription). Real-time measurement of cellular protein translation with hERG-Nanoluciferase fusions showed that initial rates of production of hERG-CM slightly exceed those of hERG-NT but that after about 4 hours this reversed with hERG-NT production increasing. Cell-free, In Vitro Translation system, which lacks the machinery to regulate RNA levels found in live cells, generated approximately 3-fold more hERG-CM protein than hERG-NT.ConclusionsOur specific synonymous modification of hERG, (with identical protein sequence) begins translation earlier with higher rates of channel production. Within the cell, however, hERG-CM is more rapidly degraded than hERG-NT which ultimately controls the final steady-state channel production. Future experiments investigating factors that influence the transcription and translation of hERG in induced pluripotent stem cell-derived cardiomyocytes to investigate these effects in a more physiological environment are in progress.