Identification of RBM5 and RBM10-dependent splicing events in 293Flp-In cells.

RBM5 and RBM10 are RNA-binding proteins and splicing regulators. These two proteins are putative paralogs in mammalian cells, sharing common domain organization and extensive protein sequence similarity, but their RNA-binding preferences differ. We developed a sensitive system to identify splicing events regulated by RBM5 and/or RBM10, deleting all RBM5 alleles in 293Flp-In cells, and reducing the expression of RBM10, which is partially rescued by activation of a cryptic exon (CE) in one of the RBM10 alleles. RBM5 or RBM10 transgenes were then introduced in these RBM5-null, RBM10 mutant cells (RBM5-/-;RBM10-/CE), allowing controlled protein expression, induced with Doxycycline. Using this system we identify thousands of RBM5 and RBM10-regulated cassette exons, and note a substantial overlap between the two sets. CRISPR-Cas9 gene deletion, followed by isogenic insertion of tetracycline-inducible transgenes.