Fig. 2. Genome-wide mapping of RNA interactions for wild-type and S34F mutant U2AF1.

a Schematic illustration of U2AF1 iCLIP. IP, immunoprecipitation; RNase, ribonuclease. Doxycycline-inducible FLAG-HA-tagged wild-type and S34F-mutant U2AF1 plasmids were transfected into HCC78 cells. Induced lysates were purified on anti-Flag-M2 agarose beads followed by a series of wash steps to specifically elute FLAG peptide-containing complexes and recaptured with anti-HA agarose. Standard iCLIP steps were subsequently performed to generate deep sequencing libraries. b Western blots were performed to confirm doxycycline induction of FLAG-HA-U2AF1 wild-type and S34F mutant constructs utilizing anti-HA and anti-U2AF1 monoclonal antibodies. Percent of HA-tagged U2AF1 of total U2AF1 is shown. c Autoradiogram of ³²P-labeled RNA crosslinked to Flag-HA-U2AF1 trimmed with two different concentrations of RNase A. RNA-protein complexes are seen in the purifications from Flag-HA-U2AF1 cells but not from the parental control cell line, HCC78. d Genomic distribution of U2AF1 iCLIP reads. lncRNA, long noncoding RNA. Wild-type and mutant U2AF1 iCLIP reads were annotated to known repetitive and non-repetitive regions of the human genome with percentage of total iCLIP reads shown. e Binding distribution of wild-type and S34F mutant U2AF1 iCLIP reads. UTR untranslated region, CDS coding sequence; 3′ SS.