CircCDYL Association With hnRNPL Modulates CDYL Isoform Switching in Breast Cancer Cells

: Circular RNAs (circRNAs) are covalently closed back-splicing products involved in the regulation of different cellular processes, and their dysregulation has been frequently observed in cancer cells. CircCDYL, a circRNA derived from the back-splicing of CDYL exon 4, has an emerging role in breast cancer (BC) biology. In this study, we investigated the role of circCDYL in modulating alternative splicing (AS) and isoform switching in MCF-7 BC cells. The circRNA profiling in MCF-7 showed circCDYL as the most abundant circRNA, with an expression increasing upon Estrogen Receptor α (ERα) silencing. RNA-Sequencing analysis of circCDYL knock-down cells revealed significant alterations in the splicing pattern, with over 2900 AS events significantly affected. Through RNA immunoprecipitation and RNA pull-down assays, we found evidence of an association between circCDYL and the splicing factor hnRNPL. To explore the consequences of this association, we compared the RNA-Sequencing of hnRNPL-silenced cells, unraveling 96 overlapping AS events accompanied by a switching usage of 223 isoforms, including those of CDYL. The self-loop regulation of circCDYL on its host gene was confirmed by isoform-specific qRT-PCR, observing that it was primarily dependent on an alternative promoter usage, rather than an AS regulation. Accordingly, epigenetic changes at CDYL alternative promoters were confirmed in circCDYL and hnRNPL knockdown cells. The confirmation of a chromatin occupancy of hnRNPL and ERα at CDYL-regulated promoters supported the role of these proteins in CDYL regulation. Our results support a synergic activity of circCDYL and hnRNPL in the regulation of AS and promoter usage in BC cells.