eCLIP in immortalized human mammary epithelial cells

The epithelial-to-mesenchymal transition (EMT) contributes to tumor heterogeneity and has been implicated in tumor initiation and metastasis. To systematically identify genes involved in EMT, we performed a genome-scale expression screen in human mammary epithelial cells and found a striking enrichment in RNA splicing factors. In particular, the RNA-binding proteins QKI and RBFOX1 were necessary and sufficient to promote EMT and stem-like states. Among the transcripts cooperatively regulated by both factors, we found that alternative splicing of the actin-binding protein FLNB plays an essential role in the regulation of EMT. The skipping of FLNB exon 30 and the elevated expression of QKI were strongly associated with EMT gene signatures in both basal B subtype breast cancer cell lines and basal-like breast cancer patient samples. These observations demonstrate that alternative splicing regulated by QKI and RBFOX1 plays an active role in promoting EMT in basal-like breast cancers. Overall design: Immortalized human mammary epithelial cells stably overexpressing V5-tagged QKI or RBFOX1 were treated by UV crosslinking at 254nm with 400mJ/cm^2. Cells were processed as described in Van Nostrand et al (PMID: 27018577), with some slight modifications, as described in Li et al, 2017 (submitted).Immunoprecipitation was done using anti-V5 antibody (Life Technologies R96025) and for each protein, libraries were prepared from two replicates plus one size-matched input control for sequencing on the NextSeq 500 (single-end, 75bp).