Structuring of the 3′ Splice Site by U2AF65

Recognition of the 3′ splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA (pre-mRNA) in a multiprotein splicing commitment complex. It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3′ splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models. We have examined the interaction of U2AF65 with an RNA representing the 3′ splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron-EDTA. These studies, combined with an analysis of extant high resolution x-ray structures of protein·RNA complexes, suggest a model whereby U2AF65 bends the pre-mRNA to juxtapose reactive functionalities of the pre-mRNA substrate and organize these structures for subsequent spliceosome assembly.