Systematic loss of function screens identify pathway specific functional circular RNAs

Abstract

Circular RNAs (circRNAs) are covalently closed single stranded RNAs that are produced by RNA back-splicing. A small number of circRNAs have been implicated as functional, however, we still lack systematic understanding of cellular processes and signalling pathways that are regulated by circRNAs. A major gap in understanding circRNA function is the ability to define pathways that are regulated by circRNAs. Here, we generated a pooled shRNA library targeting the back-splice junction of 3,354 human circRNAs that are expressed at low to high levels in humans. We used this library for loss of function proliferation screens in a panel of 18 cancer cell lines from four tissue types that harbour mutations leading to constitutive activity of defined pathways. Using this dataset, we identify context specific and non-specific circRNAs. We validated these observations with a secondary screen and uncovered a role for circRERE, a cell essential circRNA that regulates ferroptosis. Furthermore, we characterised the functional roles of pathway-specific circRNA, circSMAD2, a novel regulator of the WNT pathway and circMTO1, a regulator of MAPK signalling in a PTEN dependent manner. Our work sheds light on molecular pathways regulated by circRNAs and provides a catalogue of circRNAs with a measurable function in human cells.