PO-01-145 DYNAMIC UTR COMPOSITION IMPACTS RNA BINDING PROTEIN COMPLEXING TO CONTROL TRANSLATION AND LIMIT CARDIAC GAP JUNCTION FORMATION DURING STRESS

Abstract

Translational control has emerged as a critical yet dynamic process, representing one of the most rapid means by which the cardiomyocyte can alter its proteome. Several key cardiac mRNAs, including connexin43 (Cx43, gene name GJA1) are understood to undergo alternative translation initiation with resulting products (ie, GJA1-20k) impacting cardiac function. We previously found alterations to GJA1 mRNA untranslated region (UTR) length occur in aged and stressed heart tissue, suppressing internal translation of GJA1-20k, and limiting Cx43 gap junction formation. Having therefore identified dynamic mRNA cis element-based regulation of Cx43/GJA1-20k translation, we next asked what trans-acting factors are at play to alter GJA1 mRNA translation during stress. The IMP family of RNA binding proteins (RBPs) regulate alternative and canonical translation initiation of several genes, which led us to investigate how they may impact GJA1 mRNA. Using GJA1 as a model RNA, we test the hypothesis that alternative transcription start site usage controls inclusion of cis-acting elements in the mRNA 5’UTR affecting RBP binding and regulation of translation. Pathological hypertrophy was induced by isoproterenol deliverey to adult mice via implanted minipumps. Mouse models are complemented with matured human IPSC-derived cardiomyocytes, primary mouse ventricular cardiomyocytes, exposed to chronic hypoxia or TGF-β in vitro. Single molecule fluorescence in situ hybridization (smFISH) coupled with super-resolution microscopy localize GJA1 mRNA and RBPs. Hypertrophic hearts show reduced GJA1-20k and IMP1 expression. Using smFISH to label GJA1 mRNA, we find IMP1 complexes with GJA1 mRNA predominantly in comparison to IMP2 and 3, and that this complexing is reduced during stress, concomitant with loss of GJA1-20k internal translation. Biochemical assays confirm GJA1 mRNA/IMP1 binding by RNA-IP and synthetic reduction of IMP1 expression by RNAi or CRISPR/Cas9 alters GJA1 mRNA localization, reduces internal translation of GJA1-20k, and limits gap junction formation. We find comparable results during hypoxia in primary mouse cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes. Dynamic GJA1-5’UTR composition therefore represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation by RBPs such as IMP1, disruption of intercellular communication during stress, and arrhythmogenesis.