Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence.

长链非编码RNA（long non-coding RNAs, ncRNAs）是基因表达与细胞命运的核心调控因子。INK4基因座编码细胞周期阻滞所必需的肿瘤抑制蛋白p15INK4b、p16INK4a与p14ARF，且这些蛋白的表达在细胞衰老过程中会显著升高。ANRIL是一种反义于p15基因的ncRNA。在增殖细胞中，ANRIL通过招募多梳蛋白家族（Polycomb-group proteins, PcG）沉默INK4家族基因，从而抑制细胞衰老进程。在复制性衰老及RASval12癌基因诱导的衰老（oncogene-induced senescence, OIS）模型中，ANRIL与多梳蛋白的表达水平均会下调，进而解除INK4基因的沉默。本研究在RAF1介导的OIS模型中却发现，ANRIL的表达反而上调，这一现象主要源于其RNA稳定性的提升。基于此，我们针对环状ANRIL剪接变体展开了探究——环状RNA属于稳定性较强的RNA物种。我们发现，在RAF1、MEK1与BRAF介导的多种OIS模型中，两种环状ANRIL的表达水平均会上调。在增殖细胞中，这两种环状ANRIL会沉默p15基因的表达；而在RAF1介导的OIS模型中，它们反而会促进p15、p16与p14ARF基因的完全诱导表达。对其中一种环状ANRIL的进一步分析显示，它可与多梳蛋白结合，并分别降低多梳蛋白EZH2（Enhancer of Zeste Homolog 2）在p15与p16基因启动子区域的定位水平，以及组蛋白H3第27位赖氨酸三甲基化（H3K27me3）的修饰水平。我们提出假说：在RAF1介导的OIS过程中，多梳蛋白与环状ANRIL剪接变体的比例变化，会使得这些变体从p15基因的沉默因子转变为所有INK4家族基因的激活因子。本研究数据表明，ANRIL的表达调控依赖于衰老诱导剂的类型，同时也凸显了环状ANRIL在INK4基因表达调控与细胞衰老过程中的关键作用。