Abstract 15731: Rpl13a Small Nucleolar RNAs Promote Atherosclerosis and Oxidative Stress

Reactive oxygen species (ROS) exacerbate atherosclerosis (athero). ROS levels are elevated by specific non-coding, small nucleolar (sno) RNAs encoded within introns of the gene. We therefore tested the hypothesis that these snoRNAs promote athero, using “snoKO” mice deficient in snoRNAs, but not in itself. ROS levels assessed by CellROX Orange were 35% lower in snoKO than snoRNA aorta frozen sections ( <0.01). After 14 wk on Western diet, female snoKO/ unexpectedly showed total cholesterol levels 20% higher than mice in (1,046 vs 869 mg/dl, <0.05). Despite this, neointimal lesions in brachiocephalic artery (BCA) cross-sections were 50% smaller in snoKO/ than in mice, and lumen size was 45% larger (both <0.01, n=8/group). Similar data were obtained in males: snoKO/ mice had 40% smaller BCA lesion areas ( <0.02, n=8/group). After being stained for cholesteryl ester with BODIPY, for ACTA2 by immunofluorescence and for DNA (Hoechst), BCAs from female snoKO/ mice (n=9) demonstrated 50% less foam cell-positive and 95% more ACTA2 area, 40% less necrotic core area, and a 60% lower prevalence of ACTA2 foam cells ( <0.05 for each). Thus, snoRNAs promote vascular ROS and athero. Assessed by MitoSOX Red, ROS levels were 25% lower in snoKO than WT M1-polarized bone marrow-derived Mϕs in vitro (n=3, p<0.05). To identify mechanisms linking the snoRNAs to ROS and athero, we performed LC-MS/MS on WT and snoKO aortic SMCs. COX4I2 was expressed 5.7-fold higher in snoKO than WT SMCs by MS/MS, and 2.5-fold higher in snoKO SMCs by immunoblot (n=3/group, <0.05). As part of mitochondrial complex IV, COX4I2 lowers cellular ROS levels. We used CRISPR/Cas9 to create 293T cells lacking either the -snoRNA or the irrelevant snoRNA . With mRNA from these cells we performed reverse transcription at low [dNTP] followed by qPCR (RTL-P) for . Inversely proportional to the degree of mRNA 2’- -methylation (mediated by snoRNAs), the RTL-P efficiency was 4-fold higher in -knockout than control cells (3 clones/genotype, <0.01). Thus, mRNA appears to be regulated by -snoRNA-guided 2’- -methylation in a manner that could link snoRNAs, vascular ROS, and athero.

Duke Scholars

Author Christopher Lee Holley Medicine, Cardiology