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.
The ANRIL ncRNA is produced as an antisense RNA to the p15-encoding gene from the INK4 locus, which encodes three tumor suppressor proteins required for cell cycle arrest, p15 INK4b , p16 INK4a and p14 ARF . In proliferative cells, ANRIL prevents cellular senescence by repressing the expression of INK4 genes through Polycomb protein recruitment. Decreased expression of ANRIL in senescence is thought to relieve this repression. Here, we observe in various models of oncogene-induced senescence that senescence induction is accompanied by an increase in ANRIL expression, including the increase of circular ANRIL species. Circular ANRIL species repress p15 gene expression in proliferative cells as previously described for ANRIL. However, in RAF1 oncogeneinduced senescent cells, circular ANRIL isoforms are required for full induction of p15, p16 and p14 ARF gene expression. They interact with Polycomb group proteins and regulate H3K27me3 at the p16 gene promoter. Hence, we propose a mechanism relying on changes in the ratio between circular ANRIL and Polycomb proteins by which circular ANRIL species switch from being repressors of p15 expression to activators of INK4 gene expression during RAF1-induced senescence. Our data highlight the functional importance of the ratio between ncRNAs and their interacting effectors in the control of cell fate progression.
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