Abstract 16994: RNA Editing Controls Gene Expression in Atherosclerotic Heart Disease by Enabling HuR-mediated Posttranscriptional Regulation

Abstract

Background: Adenosine to inosine (A-to-I) RNA editing is catalysed by ADARs (adenosine deaminases acting on RNA) and is an important posttranscriptional regulator of RNA metabolism. Although RNA editing is essential for life, its role in cardiovascular disease is unknown. Methods and Results: Next generation RNA-sequencing (RNA-seq) of human endothelial cells revealed that ADAR1 edits almost 25% of endothelial transcriptome, regulates gene expression (135 transcripts were at least 2-fold regulated after ADAR1 knockdown) and controls endothelial cell function. Among the highest edited transcripts is cathepsin S (CTSS), an extracellular matrix degradation enzyme with an established role in cardiovascular and inflammatory diseases. The number of edited nucleotides in the 3’UTR of CTSS mRNA was increased under hypoxic conditions and was strongly associated with increased CTSS expression (r=0.971, P=0.029). Most importantly, absence of RNA editing resulted in a 3-fold decrease of CTSS mRNA expression under hypoxic conditions. RNA-seq analysis of 8 patients with coronary heart disease (CHD) and 4 healthy subjects revealed that A-to-I RNA editing of CTSS 3’UTR is significantly increased in patients with CHD compared to control healthy subjects (P<0.001). Most importantly, a strong association was observed between RNA editing in the CTSS 3’UTR and mRNA expression in our cohort (r>0.7, P<0.001). Mechanistically, RNA-immunoprecipitation, mRNA stability, luciferase reporter assays, mutagenesis experiments and NMR spectroscopy revealed that silencing of ADAR1 completely inhibited RNA editing of the 3’UTR of CTSS mRNA, which consequently affected CTSS RNA secondary structure, HuR binding to the CTSS 3’UTR and finally CTSS mRNA stability and protein expression (P<0.001 for all). Taken into consideration that 82% of edited genes in 3’UTR are enriched for HuR binding sites, RNA editing may critically regulate HuR-mediated posttranscriptional regulation and thus gene expression. Conclusion: This study suggests a novel primate-specific mechanism that may account for the increased cathepsin S expression in ischemic/inflammatory diseases and it may serve as a prototypical example for the evaluation of RNA-based mechanisms in health and disease.