Figure 6.

The RS domain of SF2/ASF is required for splicing of a substrate with a weak polypyrimidine tract. (A) In vitro splicing of β-globin pre-mRNA with an improved (βPy↑ ), a weakened (βPy↓ ), or no (βPyΔ) polypyrimidine tract in HeLa nuclear extract (lanes 1,7,13), S100 extract alone (lanes 2,8,14), or S100 extract complemented by 4 or 8 pmole of recombinant SF2/ASF (lanes 3,4,9,10,15,16) or ΔRS (lanes 5,6,11,12,17,18). (B) βPy↑ , βPy↓ or βPyΔ pre-mRNAs were incubated in HeLa nuclear extract (lanes 1,5,9), S100 extract alone (lanes 2,6,10), or S100 extract complemented by 8 pmole of either SF2/ASF (lanes 3,7,11) or ΔRS (lanes 4,8,12) under splicing conditions for 20 min. The reactions were then irradiated with UV light and digested with RNases A and T1. After immunoprecipitation with anti-U2AF⁶⁵ monoclonal antibody, the cross-linked U2AF⁶⁵ was detected by 12% SDS-PAGE and autoradiography. The sequence at the 3′ end of each intron is shown. The branchpoint A is shown in bold, and the four nucleotide changes in βPy↓ relative to βPy↑ are indicated.