Genome-wide Analysis Of U2AF Functions In Pre-mRNA Splicing

U2AF, a heterodimer consisting of a65kDa and a35kDa subunit, is well established for its role in defining functional3'splice site in pre-mRNA splicing, but multiple critical problems are still outstanding. First of all, it has been unclear whether it is universally required for3'splice site recognition in mammalian genomes. U2AF has also been implicated in regulated splicing, but the mechanism is only partially understood. Importantly, the functional impact of their cancer-associated mutations is still unclear. Through genome-wide analysis of U2AF-RNA interactions (CLIP-seq), coupld with RNA-seq and RASL-seq data analysis, we report that U2AF has the capacity to define-88%of functional3'splice sites in the human genome. Numerous U2AF binding events also occur in other genomic locations and minigene analysis suggests that upstream intronic binding events interfere with the immediate downstream3'splice site associated with either the alternative exon to cause exon skipping or competing constitutive exon to induce inclusion of the alternative exon. We demonstrate that the U2AF heterodimer function together in splicing regulation and U2AF35protein level is maintained by a post-translational mechanism. We further demonstrate partial functional impairment with mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.