RNA secondary structure modulates the co-translational association of hERG1a and 1b mRNAs

Cardiac IKr is an important repolarizing potassium current of the human ventricular action potential. It is conducted by heteromeric assemblies of the human ether-à-go-go-related gene (hERG1) 1a and 1b subunits. These subunits are encoded by alternate transcripts of the hERG1/KCNH2 gene and differ only in their amino-terminal regions. hERG1a/1b heteromerization is vital for normal CM function, as the imbalance of the subunit's expression and/or function results in cellular pro-arrhythmic behaviors. hERG1a/1b assembly is mediated by the co-translational association of the encoding mRNAs in HEK293 cells, cardiomyocytes derived from human induced pluripotent stem cells, and human myocardium. New evidence suggests that neither 1a nor 1b proteins are required for the co-translational complex assembly. Thus, we hypothesize that hERG1a and 1b mRNAs’ physico-chemical properties, especially their structure, contribute to their co-translational association. In an in vitro reconstitution assay, hERG1a and 1b mRNA transcribed and labeled with Cy3 and Cy5, respectively, undergo phase separation in isolation. When mixed, the two transcripts modulate each other's morphology as represented by size, shape, and area covered, suggesting they interact. Synonymous mutations predicted to alter hERG1a secondary structure, without affecting channel function, reduce hERG1a and 1b interaction in vitro. Confocal live-cell imaging in HeLa cells transfected with labeled hERG1a and 1b mRNA show condensate morphologies consistent with those seen in vitro. Co-transfection of the in-vitro labeled mRNAs results in spatial overlap of the respective signals as well as translation of hERG1b protein apparently within the same macromolecular complexes, indicating they are translational. In addition, the findings indicate that co-transcription of the mRNAs is not required for their co-translational association. These results suggest that 1a and 1b mRNA physico-chemical properties modulate their association and subsequent co-translation in live cells.