The carboxyl terminus of vertebrate poly(A) polymerase interacts with U2AF65 to couple 3 '-end processing and splicing

Although it has been established that the processing factors involved in pr e-mRNA splicing and 3'-end formation can influence each other positively, t he molecular basis of this coupling interaction was not known. Stimulation of pre-mRNA splicing by an adjacent cis-linked cleavage and polyadenylation site in HeLa cell nuclear extract is shown to occur at an early step in sp licing, the binding of U2AF 65 to the pyrimidine tract of the intron 3' spl ice site. The carboxyl terminus of poly(A) polymerase (PAP) previously has been implicated indirectly in the coupling process. We demonstrate that a f usion protein containing the 20 carboxy-terminal amino acids of PAP, when t ethered downstream of an intron, increases splicing efficiency and, like th e entire 3'-end formation machinery, stimulates U2AF 65 binding to the intr on. The carboxy-terminal domain of PAP makes a direct and specific interact ion with residues 17-47 of U2AF 65, implicating this interaction in the cou pling of splicing and 3'-end formation.