Abstract Body (Do not enter title and authors here): Introduction: Despite therapeutic advances, atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death in industrialized nations, highlighting the need for precision medicine. ADAR1-mediated RNA editing has emerged as a key regulator in vascular disease, yet its interaction with the RNA-binding protein HuR, potentially governing Cathepsin expression, remains poorly understood.

Methods: Mechanistic insights into ADAR1-mediated Alu A-to-I RNA editing and its interplay with HuR were derived from RNA sequencing, editing analyses, gain- and loss-of-function studies, stability assays, HuR iCLIP, luciferase reporter assays, and RIP following ADAR1 siRNA-mediated knockdown in primary human endothelial cells. CTSK and ADAR1 expression was quantified by qRT-PCR in peripheral blood mononuclear cells (n=367) and carotid plaques (n=35) from individuals with and without ASCVD. Subclinical atherosclerosis and its progression were assessed by ultrasound and prospectively followed for MACE.

Results: Endothelial mRNA sequencing identified two significantly downregulated Cathepsins upon ADAR1 knockdown, including CTSK, a protease linked to atherosclerotic plaque progession and instability. CTSK was extensively edited within Alu regions in intron 5. Silencing of ADAR1 led to a twofold decrease in mature CTSK mRNA and accumulation of pre-mRNA, while overexpression reversed this. HuR binding motifs were near editing sites, and HuR knockdown or overexpression mirrored ADAR1 effects on CTSK processing. Actinomycin D treatment confirmed that knockdown of either ADAR1 or HuR reduced CTSK mRNA stability. Luciferase, iCLIP, and RIP assays confirmed HuR’s interaction with intronic edited CTSK regions—lost without editing (all P < 0.05). Clinically, CTSK expression strongly correlated with ADAR1 and HuR levels in ASCVD patients (r = 0.71 and 0.82, P < 0.001) and plaques (r = 0.81 and 0.86, P < 0.001). CTSK expression was independently associated with ASCVD (OR = 1.84, highest vs. lower tertiles), elevated C-reactive protein (>3 mg/dL), increased carotid maximum wall thickness (>1.39 mm; OR = 4.44), and multivessel disease (OR = 4.09) (all P < 0.05). Elevated CTSK also predicted accelerated subclinical atherosclerosis progression and higher MACE incidence over 48 months.

Conclusion: Intronic Alu-mediated RNA editing modulates HuR-dependent pre-mRNA processing, revealing a novel regulatory layer in ASCVD with implications for precision medicine.