Abstract 16931: Alternate UTR Usage Regulates Connexin43 mRNA Translation Limiting Gap Junction Formation During Stress

Abstract

Introduction: Connexin43 (Cx43, Gja1 ) gap junctions enable electrical coupling of cardiomyocytes necessary for heart function. Pathological disruption of Cx43 expression or localization results in the arrhythmias of sudden cardiac death. Investigation of Cx43 gap junction biology has focused on transcription, vesicular trafficking, and post translational modifications. The importance of translational regulation however is highlighted by the recent discovery that Cx43 mRNA undergoes internal translation initiation to yield truncated protein isoforms, including the predominant GJA1-20k isoform capable of regulating gap junction formation. We have found that TGF-β signaling, a hallmark of cardiac stressors including hypertrophy, suppresses GJA1-20k expression to limit gap junction formation. The mechanisms regulating Cx43 mRNA internal translation in response to cellular stress are unknown but are critical in harnessing this biology for therapeutic restoration of electrical coupling in diseased hearts. We hypothesize that altered Gja1 -5’UTR usage promotes translation of full length Cx43 over internal translation products resulting in reduced gap junction formation during stress. Methods: Rapid amplification of cDNA ends was used to identify Gja1- 5’UTR isoforms following TGF-β exposure or hypoxia, and Western blotting was used to monitor Gja1 translation. Identified Gja1 -5’UTR variants were transfected into Cx43 knockout cells to assess translational potential. Luciferase assays assessed potential IRES activity within Gja1 mRNA in relation to specific 5’UTR isoforms. Results: TGF-β or hypoxia significantly increase translation of full length Cx43 over Gja1-20k correlating with an increase in truncated Gja1 -5’UTRs. Sequenced UTR isoforms map to genome wide transcription start sites and transfection of Gja1 transcripts with truncated 5’UTRs is sufficient to reduce Gja1-20k translation, phenocopying stress models. Conclusions: Alteration of Gja1 -5’UTR composition represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation to fine tune intercellular communication during stress.