The long non-coding RNA AS-Trdn controls the balance of triadin isoform expression and cardiac excitation-contraction coupling

Abstract

The molecular function of long non-coding RNAs (lncRNAs) has become an emerging scientific field focusing on various molecular pathways across species. One specific subclass known as antisense lncRNAs, was defined by its genomic localization antisense to one or more protein-coding genes. In numerous instances, these lncRNAs play a crucial role in cis-regulation by modulating the expression and splicing of the associated sense protein-coding gene. In the present study, the functional significance and the physiological implications of the cardiomyocyte-specific antisense lncRNA AS-Trdn were investigated in mus musculus as well as human cells. The expression of AS-Trdn in cardiac tissue leads to a shift in triadin isoform expression, replacing the long skeletal muscle triadin with the short cardiac specific isoform. Employing various NGS approaches and CRIPR/Cas9 based genomic interventions to elucidate the molecular mechanism, this study demonstrates that AS-Trdn regulates triadin splicing and alternative polyA usage in-cis through a RNA Polymerase II transcriptional interference. Investigations involving the loss of single exons and the overexpression of the spliced transcript indicate no alterations in triadin isoform abundance. However, endogenous overexpression in differentiated satellite cells results in a preference for the skeletal muscle isoform. The conserved underlying molecular mechanism is shown to rely on the AS-Trdn transcription, inducing sequence-dependent transcriptional pausing, wherein the antisense polymerase acts as a Roadblock for the opposing polymerase. Although an R-loop dependent transcription termination is postulated due to high GC-content and m6a deposition within a specific region, confirmation of the occurrence of an RNA:DNA hybrid is pending. Physiologically, AS-Trdn and the consequent expression of cardiac triadin is essential for a regular calcium cycling, normal heart rate and a accurate response to β-adrenergic signalling. Through isoform specific interaction studies, it is revealed that skeletal muscle triadin extensively binds to the microtubule and actinin cytoskeleton resulting in significant deformation of the cardiac dyad structure compared to the healthy situation. The subsequent increase in calcium handling and heart rate leads to a commencing dilated cardiomyopathy in ageing mice, characterized by decreased cardiac function, reduced cardiomyocyte cross-sectional area and an overall increase in heart size. Consequently, AS-Trdn and triadin emerge as pivotal regulators of cardiac contraction and cardiac dyad structure, influencing both health and disease.