Fig 1: U2AF65 associates with PPTs of aCP-enhanced exons in the absence of its cognate binding site. (A) Enrichments of SF3B2–145 and U2AF65 in aCP1 and in aCP2 co-immunoprecipitates. K562 cell extracts were prepared in the presence of RNase A and subjected to immunoprecipitation with isotype-specific antisera to aCP1 and aCP2 in parallel with IP with preimmune serum (PI). Each immunoprecipitate was resolved by SDS-PAGE and then probed (Western blot) with antibodies specific to two splicing factors; SF3B2 and U2AF65. (B) Discordance between the distributions of cognate U2AF65 binding sites and U2AF65 occupancy in the regions 5' to aCP-enhanced exons. K562 U2AF65 eCLIP reads (‘U2AF65 binding density’; blue curves) are displayed upstream and downstream of the three groups of cassette exons; aCPs-enhanced, aCPs-repressed and a defined background set (as defined in Figure 1C and D). The data reveal pronounced occupancy of U2AF65 upstream of all three groups (blue curves). The distribution of the corresponding U2AF65 binding site motifs (TTTTT[C/T]C) (red curves) parallel the binding density 5' to the repressed and background exons but are dramatically under-represented 5' to the aCP-enhanced exons. These data suggest that U2AF65 may be recruited to C-rich PPTs upstream of aCP-enhanced cassette exons by an indirect mechanism.