Background: The enzyme adenosine deaminase acting on RNA-1 (ADAR1) binds to double-stranded RNAs inducing the deamination of adenosine to inosine (A-to-I), a process that is called RNA editing. We have previously reported that ADAR1 expression is increased in atherosclerosis controlling cathepsin S mRNA stability. Whether RNA editing controls other cathepsins in atherosclerosis is not known.
Methods and Results: High-throughput RNA-sequencing and RNA editing studies revealed that cathepsin K (CTSK) is extensively edited within the Alu regions of intron 5. CTSK is an extracellular matrix degradation enzyme with an established role in atherosclerosis. Silencing of ADAR1 resulted in a two-fold downregulation of CTSK mature mRNA and a two-fold upregulation of CTSK pre-mRNA expression (P<0.05 for all), indicating that ADAR1-mediated RNA editing inhibits CTSK pre-mRNA processing. In contrast, overexpression of ADAR1 led to an increase of mature processed CTSK mRNA and a decrease in unprocessed CTSK pre-mRNA (P<0.05). Given that a plethora of U-rich sites is present in intron 5, we investigated whether the RNA-binding protein HuR controls CTSK pre-mRNA processing. Indeed, silencing of HuR reduced CTSK mRNA expression by more than two-fold (P<0.05). Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) experiments using a specific antibody targeting HuR confirmed the HuR interaction with CTSK intronic edited regions. In the absence of RNA editing, HuR did not bind to the CTSK. Under pro-inflammatory (TNF-α) conditions, ADAR1 and HuR were the main regulators of CTSK mRNA expression (P<0.05). Both ADAR1 and CTSK levels are significantly upregulated in PBMC derived from patients with coronary artery disease compared to healthy subjects (P<0.05). Most importantly, CTSK levels closely correlated with the expression of both ADAR1 (r=0.707, P<0.001) and HuR (r=0.697, P<0.001).
Conclusion: RNA editing does not only affect one cathepsin, but cathepsin classes, thereby controlling the network of proteases, which may explain the profound association of ADAR1 with atherosclerosis. Given the important role of cathepsins in atherosclerosis, A-to-I RNA editing may critically link inflammation with coronary atherosclerotic disease.