CircCDYL association with hnRNPL modulates CDYL isoform switching in Breast Cancer cells
Circular RNAs (circRNAs) are covalently closed transcripts involved in the regulation of different cellular processes, and their dysregulation has been frequently observed in cancer. In this study, we investigated the role of circCDYL, a circRNA generated from the CDYL gene, in modulating alternative splicing (AS) and isoform switching in breast cancer cells. Analysis of circRNA profiles in breast cancer cells showed that circCDYL expression increased in estrogen receptor alpha (ERa)-downregulated cells, suggesting a potential link between circCDYL-mediated regulation and ERa signaling pathways. RNA-Sequencing analysis following circCDYL knock-down in MCF-7 cells revealed significant alterations in the splicing pattern, with over 2,900 splicing 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 performed AS and isoform switching analysis after circCDYL and hnRNPL silencing, revealing significant effects on AS and a weaker modulation of isoform switching events. Furthermore, we observed that circCDYL and hnRNPL modulated CDYL isoform expression, as confirmed by isoform-specific qRT-PCR analysis. Chromatin immunoprecipitation assays also indicated changes in chromatin marks at the CDYL locus upon knock-down of circCDYL and hnRNPL. These results suggested a possible association between circCDYL and hnRNPL, which contributed to the regulation of alternative splicing in breast cancer cells and may be involved in the modulation of isoform expression and chromatin dynamics of CDYL gene. Overall design: To analyze the role oh hnRNPL and circCDYL in transcriptional and splicing control, we performed total RNA-Seq on MCF-7 cells after circCDYL and hnRNPL siRNA-mediated silencing.