Circular RNAs (circRNAs), formed by non-sequential back-splicing of pre-mRNA transcripts, are a widespread form of non-coding RNA in animal cells. However, it is unclear whether the majority of circRNAs represent splicing by-products without function or are produced in a regulated manner to carry out specific cellular functions. We show that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and find that the production of over one-third of abundant circRNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT. Furthermore, by modulating QKI levels, we show the effect on circRNA abundance is dependent on intronic QKI binding motifs. Critically, the addition of QKI motifs is sufficient to induce de novo circRNA formation from transcripts that are normally linearly spliced. These findings demonstrate circRNAs are both purposefully synthesized and regulated by cell-type specific mechanisms, suggesting they play specific biological roles in EMT.

Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.