The Curriculum in Toxicology, The Lineberger Comprehensive Cancer Center, The University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America.
PLoS Genet. 2010 Dec 2;6(12):e1001233. doi: 10.1371/journal.pgen.1001233.
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, p16(INK4a), p15(INK4b), 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.
人类全基因组关联研究已经将染色体 9p21.3 上靠近 INK4/ARF（CDKN2a/b）基因座的单核苷酸多态性（SNP）与动脉粥样硬化性血管疾病（ASVD）的易感性联系起来。尽管这个基因座编码了三个特征明确的肿瘤抑制因子，即 p16(INK4a)、p15(INK4b)和 ARF，但与 ASVD 相关性最强的 SNP 位于编码长非编码 RNA（lncRNA）的基因附近，该基因称为 ANRIL（CDKN2BAS）。虽然携带动脉粥样硬化风险等位基因的个体表现出 ANRIL 和编码 INK4/ARF 转录物的表达减少，但这种远距离遗传变异如何影响 INK4/ARF 表达的机制尚不清楚。在这里，我们使用快速扩增 cDNA 末端（RACE）和下一代 RNA 测序数据集的分析，确定了多个 ANRIL 物种的结构和丰度。这些物种在原代和培养细胞中都以非常低的拷贝数存在；然而，只有包含靠近 INK4/ARF 基因座的外显子的 ANRIL 异构体的表达与 ASVD 风险等位基因相关。令人惊讶的是，RACE 还鉴定了包含非共线性 ANRIL 外显子序列的转录本，其表达也与基因型和 INK4/ARF 表达相关。这些非多聚腺苷酸化的 RNA 抵抗 RNAse R 消化，并且可以使用向外引物进行 PCR 扩增，表明它们代表可能来自 mRNA 剪接副产物的环状 RNA 结构。下一代 DNA 测序和剪接预测算法鉴定了 ASVD 风险区间内的多态性，这些多态性可能调节 ANRIL 剪接和环状 ANRIL（cANRIL）的产生。这些结果鉴定了源自 ANRIL 基因座的新型环状 RNA 产物，并表明 9p21.3 上的因果变异通过调节 ANRIL 表达和/或结构来调节 INK4/ARF 表达和 ASVD 风险。
