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

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.

Epigenetic regulation in human melanoma: past and future

“…Several isoforms of ANRIL have been reported, including various short and long isoforms, and a recently discovered circular isoform. 89 Different exons of ANRIL are differentially expressed in melanoma cell lines, and there is evidence for the existence of circular ANRIL in some of these cells. This discovery suggested that alternative splicing modifies ANRIL structure.…”

“…This discovery suggested that alternative splicing modifies ANRIL structure. 89 This mechanism has been studied in atherosclerosis, and further work is required to characterize this mechanism in melanoma. Interestingly, GWAS identified a single nucleotide variant rs1011970 (intron 9 of the ANRIL isoform with 19 exons) that is associated with melanoma risk, but only for the variant T-allele homozygote.…”

Identification of circular RNAs hsa_circ_0044235 in peripheral blood as novel biomarkers for rheumatoid arthritis

“…Recent studies have revealed that abnormal circRNAs are associated with several human diseases, such as atherosclerotic vascular disease, cancer and autoimmune disease 22, 23, 24, 25, 26. Many dysregulated expression circRNAs are demonstrated to associate with RA 16, 17.…”

“…Previous studies have shown that miRNAs and lncRNA are involved in the pathogenesis of RA and can be used as biomarkers for the diagnosis of RA 8, 9, 10, 11. Increasing evidence reveals that circRNAs are used widely in various physiological and pathological conditions, such as atherosclerotic vascular diseases, neurological disorders, prion infection and cancer 12, 13, 14, 15. However, little is known about the roles of circRNAs in human RA.…”

In vitro circularization of RNA

“…In fact, based on recent research in the field, circRNAs have been suggested to act as miRNA and protein sponges, to be involved in the modulation of gene expression and splicing, and to be associated with diseases initiation and progression. 4,7,[14][15][16][17][18] Thus, circRNAs have also been considered as biomarkers for diseases prognostics and diagnostics and as targets or tools for disease treatment. [19][20][21] The field of investigations into circRNA and in particular into their putative function within the complex network of regulation of gene expression and splicing is currently very active.…”