Expression of Linear and Novel Circular Forms of an INK4/ARF-Associated Non-Coding RNA Correlates with Atherosclerosis Risk

Top Cited Papers
Open Access
Publisher Website
Google Scholar
Abstract
Human genome-wide association studies have linked single nucleotide polymorphisms (SNPs) on chromosome 9p21.3 near the INK4/ARF (CDKN2a/b) locus with susceptibility to atherosclerotic vascular disease (ASVD). Although this locus encodes three well-characterized tumor suppressors, p16INK4a, p15INK4b, and ARF, the SNPs most strongly associated with ASVD are ∼120 kb from the nearest coding gene within a long non-coding RNA (ncRNA) known as ANRIL (CDKN2BAS). While individuals homozygous for the atherosclerotic risk allele show decreased expression of ANRIL and the coding INK4/ARF transcripts, the mechanism by which such distant genetic variants influence INK4/ARF expression is unknown. Here, using rapid amplification of cDNA ends (RACE) and analysis of next-generation RNA sequencing datasets, we determined the structure and abundance of multiple ANRIL species. Each of these species was present at very low copy numbers in primary and cultured cells; however, only the expression of ANRIL isoforms containing exons proximal to the INK4/ARF locus correlated with the ASVD risk alleles. Surprisingly, RACE also identified transcripts containing non-colinear ANRIL exonic sequences, whose expression also correlated with genotype and INK4/ARF expression. These non-polyadenylated RNAs resisted RNAse R digestion and could be PCR amplified using outward-facing primers, suggesting they represent circular RNA structures that could arise from by-products of mRNA splicing. Next-generation DNA sequencing and splice prediction algorithms identified polymorphisms within the ASVD risk interval that may regulate ANRIL splicing and circular ANRIL (cANRIL) production. These results identify novel circular RNA products emanating from the ANRIL locus and suggest causal variants at 9p21.3 regulate INK4/ARF expression and ASVD risk by modulating ANRIL expression and/or structure. Unbiased studies of the human genome have identified strong genetic determinants of atherosclerotic vascular disease (ASVD) on chromosome 9p21.3. This region of the genome does not encode genes previously linked to ASVD, but does contain the INK4/ARF tumor suppressor locus. Products of the INK4/ARF locus regulate cell division, a process thought to be important in ASVD pathology. We and others have suggested that genetic variants in 9p21.3 influence INK4/ARF gene expression; however, the mechanisms by which these distant polymorphisms (>100,000 bp away) influence transcription of the locus is unknown. The ASVD–associated genetic variants lie within the predicted structure of a non-coding RNA (ncRNA) called ANRIL. Based upon recent work suggesting that other ncRNAs can repress nearby coding genes, we considered the possibility that ANRIL structure may regulate INK4/ARF gene expression. Coupling molecular analysis with state-of-the-art sequencing technologies in a wide variety of cell types from normal human donors and cancer cells, we found that ANRIL encodes a heterogeneous species of rare RNA transcripts. Moreover, we identified novel, circular ANRIL isoforms (cANRIL) whose expression correlated with INK4/ARF transcription and ASVD risk. These studies suggest a new model wherein ANRIL structure influences INK4/ARF expression and susceptibility to atherosclerosis.