Post-transcriptional RNA processing is a fundamental biological process that is important for regulation of most aspects of life. Circular RNA (circRNA), produced from precursor mRNA back-splicing, was shown to be abundant across diverse cell types and play vital roles in cancer progression. However, the absence of tools to systematically perturb circRNAs has hindered the study of circRNA function. The primary objective of our study is to develop an approach that enables specific knockdown of circRNA using short hairpin RNA (shRNA) to systematically identify circRNA dependencies in specific signalling pathways.
We generated a pooled shRNA library targeting the back-splice junction of 3,354 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 lineages that harbour mutations leading to constitutive activity of defined pathways. Our data has identified context specific and non-specific circRNAs. We validated these observations in a secondary screen and focused on circMTO1 that we identified as a regulator of MAPK signalling in a PTEN dependent manner.
CircRNAs are poorly characterised non-coding RNAs which play important roles in the development of human diseases. Our work sheds light on molecular pathways regulated by circRNAs and also provides a powerful framework for studies utilising circRNAs as therapeutic targets in specific disease treatment.