Figure 4.

Binding properties of the 65-kDa protein and its target site within the NRE. (A) HeLa cell nuclear proteins were UV-crosslinked to probes corresponding to the 5′ half of the NRE (probe 5′) with four 5′ splice site homologies, or the 3′ half of the NRE (probe 3′) with a G+U-rich region with homology to a U2AF⁶⁵ binding site. Poly(U) denotes nuclear extracts depleted by preincubation with poly(U) Sepharose in the presence of 2 M KCl. Unlabeled RNA corresponding to the middle section of the NRE (m, see Fig. 2) was used as competitor. (B) Reconstitution experiment with U2AF⁶⁵. The indicated amounts (in micrograms) of partially purified, bacterial protein extract containing U2AF⁶⁵ were added to HeLa cell nuclear extracts that were depleted of 65-kDa protein binding activity by preincubation with poly(U) Sepharose in 2 M KCl, probe N was used (Fig. 2).