ABSTRACT

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15^INK4b, p16^INK4a and p14^ARF 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 p14^ARF 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.

摘要

长链非编码 RNA (ncRNA) 是基因表达和细胞命运的主要调节因子。的INK4基因座编码肿瘤抑制蛋白p15基因^INK4B，P16 ^INK4A和P14 ^ARF需要细胞周期停滞和衰老过程中其表达增加。ANRIL是p15基因的 ncRNA 反义序列。在增殖细胞中，ANRIL通过募集 Polycomb 组蛋白抑制INK4基因来防止衰老。在复制和 RASval12 致癌基因诱导的衰老 (OIS) 模型中，ANRIL和 Polycomb 蛋白的表达降低，从而允许INK4去压抑。在这里，我们在 RAF1 OIS 模型中发现ANRIL表达反而增加，特别是由于稳定性增加。这导致我们寻找环状ANRIL同种型，因为环状 RNA 是相当稳定的物种。我们发现两个圆形ANRIL在几个 OIS 模型（RAF1、MEK1 和 BRAF）中的表达增加。在增殖细胞中，它们抑制p15表达，而在 RAF1 OIS 中，它们促进p15、p16和p14 ^ARF表达的完全诱导。对这些环状ANRIL之一的进一步分析表明，它与 Polycomb 蛋白相互作用并降低 EZH2 Polycomb 蛋白定位和 H3K27me3 在分别为p15和p16启动子。我们建议 Polycomb 蛋白和环状ANRIL异构体之间比例的变化允许这些异构体从p15基因的阻遏物转换为RAF1 OIS中所有INK4基因的激活物。我们的数据显示ANRIL表达的调节取决于衰老诱导剂，并强调了环状ANRIL在调节INK4基因表达和衰老中的重要性。