Il network di circCDYL nella regolazione del suo gene ospite, CDYL, in linee cellulari di cancro al seno

Circular RNAs (circRNAs) are a new class of mostly non-coding molecules characterized by a covalently closed structure. For a long while considered to be splicing errors, in recent years they have been described as stable and tissue-specific molecules. Here we focus the attention on circCDYL, a circular RNA arising from one exon of the human gene CDYL and reported to be highly expressed in different cancer cell lines. Interestingly, we found that circCDYL can regulate isoform switching of its host gene in breast cancer cells MCF-7 and MDA-MB-231. CircCDYL silencing increases the levels of CDYL-204, an isoform which normally is not expressed, and does not change the widely expressed CDYL-203 isoform. CDYL-204 isoform lacks the exon involved in circCDYL synthesis and the Chromodomain, which, instead are both present in CDYL-203. In this structural context, CDYL-204 still preserves the interaction site with EZH2, a PRC2 catalytic subunit altering genes expression by H3K27me3 deposition. In order to understand the molecular mechanism underpinning isoform selection by circCDYL, we used two approaches: firstly, we focus on epigenetic level and then we investigated circCDYL interactions. We explored the epigenetic profile of the isoform-specific promoters using ChIP-qPCR and we found that circCDYL silencing changes H3K27Ac enrichment, epigenetic mark for active gene, at the promoters of CDYL-203 and 204 isoforms. Studies on circCDYL-RBP interactions were performed using bioinformatic tools and RNA pull-down followed by high resolution mass-spectrometry. We selected and validated two putative circCDYL’s interactors, hnRNP-H and hnRNP-L, heterogenous nuclear ribonucleoproteins (hnRNPs) involved in the splicing mechanism in healthy and cancer tissue. We hypothesize that circCDYL can recruit and sequester RBPs regulating the splicing events of a chromatin remodeling factor. The circCDYL knock-down may release RBPs taking to the selection of isoform-specific factor. This unknown factor may work as chromatin remodeler of CDYL-204 promoter inducing its expression.