Intronic ALU RNA editing couples mRNA stability with pre-mRNA processing of inflammatory gene expression in atherosclerosis

Background and Aims: Adenosine deaminase acting on RNA-1 (ADAR1) binds double-stranded RNAs (e.g Alu elements) and deaminates adenosine to inosine (A-to-I), a process called RNA-editing. Although most of the RNA-editing events are located in introns, the role of intronic RNA editing in gene expression and pathophysiology remains elusive. Methods: RNA-sequencing, RNA-editing studies, gain/loss-of-function assays, gene expression analysis, individual cross-linking immunoprecipitation (iCLIP) and RNA immunoprecipitation (RIP) studies in human primary endothelial cells were employed. Cathepsin K (CTSK) and ADAR1 mRNA expression were measured by qRT-PCR in peripheral blood mononuclear cells and carotid plaques from patients with cardiovascular disease (n = 91) and healthy individuals (n = 131). Results: RNA-sequencing and RNA-editing studies revealed editing events in CTSK, an inflammation-stimulated extracellular matrix degradation enzyme with an established role in atherosclerosis. CTSK is extensively edited within the Alu regions of intron 5, also enriched of HuR binding sites. Silencing of ADAR1 resulted in a 2-fold downregulation of CTSK mature mRNA and a 2-fold upregulation of pre-mRNA. ADAR1 overexpression exerted the opposite. Silencing of HuR reduced CTSK expression by >2-fold. iCLIP and RIP experiments confirmed that HuR interacts with intronic edited regions of CTSK. In the absence of RNA editing, HuR did not bind CTSK. ADAR1 and CTSK levels were significantly upregulated in patients compared to healthy subjects. CTSK levels closely correlated with the expression of ADAR1 in patients(p < 0.001, r = 0.707). Conclusions: Intronic Alu RNA-editing enables proper pre-mRNA processing by HuR, a primate-specific mechanism that plays a decisive part in inflammatory gene expression during atherosclerotic heart disease.