U1 snRNP targets an essential splicing factor, U2AF65, to the 3′ splice site by a network of interactions spanning the exon

Abstract

A description of cellular factors that govern alternative splicing of pre-mRNA is largely incomplete. In the case of the rat preprotachykinin gene, splicing of the alternative exon E4 occurs by a poorly understood mechanism in which exon selection is under the positive control of U1 snRNP. Because the binding of U1 snRNP to the 5′ splice site of E4 is coincident with the selection of the 3′ splice site of E4, this mechanism would appear to involve interactions that bridge across the exon. In this work, a UV cross-linking strategy was used to identify possible RNA-protein interactions involved in the proposed exon-bridging model. Of particular interest is a prominent 61-kD protein, p61, that binds to the 3′ splice site of E4 in a manner that is clearly facilitated by a downstream 5′ splice site and U1 snRNP particles. The identity of p61 is the essential splicing factor U2AF65, on the basis of copurification and selective binding to polypyrimidine tracts. These results indicate a model in which exon selection is positively regulated by the communication of U1 snRNP and U2AF65. That is, a natural deficiency in binding U2AF65 to the 3′ splice site that leads to exon skipping might be overcome by a mechanism in which U1 snRNP facilitates the binding of U2AF65 through a network of template-directed and exon-bridging interactions.