Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease.

The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) antisense non-coding RNA in the INK4 locus (ANRIL). ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.

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C-reactive protein (CRP), a phylogenetically highly conserved plasma protein, is the classical acute phase reactant in humans. Upon infection, inflammation, or tissue damage, its plasma level can rise within hours >1000-fold, providing an early, nonspecific disease indicator of prime clinical importance. In recent years, another aspect of CRP expression has attracted much scientific and public attention. Apart from transient, acute phase-associated spikes in plasma concentration, highly sensitive measurements have revealed stable interindividual differences of baseline CRP values in healthy persons. Strikingly, even modest elevations in stable baseline CRP plasma levels have been found to correlate with a significantly increased risk of future cardiovascular disease. These observations have triggered intense controversies about potential atherosclerosis-promoting properties of CRP. To directly assess potential effects of CRP on atherogenesis, we have generated CRP-deficient mice via gene targeting and introduced the inactivated allele into atherosclerosis-susceptible ApoE−/− and LDLR−/− mice, two well established mouse models of atherogenesis. Morphometric analyses of atherosclerotic plaques in CRP-deficient animals revealed equivalent or increased atherosclerotic lesions compared with controls, an experimental result, which does not support a proatherogenic role of CRP. In fact, our data suggest that mouse CRP may even mediate atheroprotective effects, adding a cautionary note to the idea of targeting CRP as therapeutic intervention against progressive cardiovascular disease.