Functional Significance of U2AF1 S34F Mutation in Lung Adenocarcinomas

We conducted RNA-Seq and iCLIP for dox-inducible constructs of FLAG-HA tagged U2AF1 in the wild-type and mutant forms. Overall design: The wild-type and mutant versions of U2AF1 were generated in HCC78. The goals of this study were to find differential binding and expression of wild type and mutant U2AF1. We generated stable doxycycline-inducible U2AF1 wild-type and mutant cell lines in HCC78 and induced each isoform with doxycycline. We observed that compared to its wild-type counterpart, U2AF1 S34F preferentially binds and modulates splicing of introns containing CAG trinucleotides at their 3' splice junctions. The presence of S34F caused a shift in binding at 3' splice sites, which was significantly associated with alternative splicing of skipped exons. Expression of U2AF1 S34F also induced the expression of genes involved in the epithelial-mesenchymal transition (EMT), with corresponding increases in tumor cell invasion. Finally, consistent with its RNA binding affinities, S34F preferentially induced splicing of the long over the short SLC34A2-ROS1 isoform, and overexpression of the long isoform increased invasiveness.