Figure 6.

Cooperative binding of U2AF65^RRM123 and SF1 to a 3′-splice site RNA. RNA was incubated with buffer or U2AF65^RRM123 (0.2, 0.5, 1 and 2 µM; indicated by triangles) in the absence or presence of 1.5 µM His₆-tagged SF1^2-320 or internal deletion mutants (A) or with 6.6 µM SF1 or pSF1 (B). Reaction products were separated by native PAGE and visualized by autoradiography. The migration of SF1–U2AF65–RNA complexes (closed arrowhead) and SF1–RNA complexes (open arrowhead) is indicated. (C) Role of SF1^HH and the SF1^NTD–U2AF65^UHM interaction in the formation of the 3′-splice site recognition complex. SF1^HH may establish an optimal orientation of the SF1 (KH–QUA2) and U2AF65^RRM1,2 RNA-binding subunits in the complex and thereby support cooperative RNA binding. Tandem phosphorylation of SF1 might contribute to the formation of the ternary complex by stabilizing a yet unknown interface.