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

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The <i>INK4</i> locus encodes the tumour suppressor proteins p15^INK4b, p16^INK4a and p14^ARF required for cell cycle arrest and whose expression increases during senescence. <i>ANRIL</i> is a ncRNA antisense to the <i>p15</i> gene. In proliferative cells, <i>ANRIL</i> prevents senescence by repressing <i>INK4</i> genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of <i>ANRIL</i> and Polycomb proteins decreases, thus allowing <i>INK4</i> derepression. Here, we found in a model of RAF1 OIS that <i>ANRIL</i> expression rather increases, due in particular to an increased stability. This led us to search for circular <i>ANRIL</i> isoforms, as circular RNAs are rather stable species. We found that the expression of two circular <i>ANRIL</i> increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress <i>p15</i> expression, while in RAF1 OIS, they promote full induction of <i>p15, p16</i> and <i>p14^ARF</i> expression. Further analysis of one of these circular <i>ANRIL</i> shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the <i>p15</i> and <i>p16</i> promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular <i>ANRIL</i> isoforms allow these isoforms to switch from repressors of <i>p15</i> gene to activators of all <i>INK4</i> genes in RAF1 OIS. Our data reveal that regulation of <i>ANRIL</i> expression depends on the senescence inducer and underline the importance of circular <i>ANRIL</i> in the regulation of <i>INK4</i> gene expression and senescence.