FIGURE 4.

A spliceosome regulatory complex specifically assembles on exon 7. A, unlabeled competitor RNA can titrate an exon 7-associated nuclear complex. Left panel, ³²P-labeled exon 7 RNA (0.28 pmol) was incubated in the presence (+) (lanes 2–8) or absence (−) (lane 1) of HeLa nuclear extract (NE) under splicing conditions and resolved on a native gel. Unlabeled competitor RNA was also added to the reactions as indicated. Right panel, an overexposed membrane (lanes 6–8) of the left panel is shown. B, UV light-induced cross-linking to exon 7 reveals nuclear and cytoplasmic proteins. S100 extract (containing cytoplasmic proteins) cross-linking reactions were loaded in lanes 3 and 5. The HeLa extract (containing nuclear proteins)-cross-linking reactions were loaded in lanes 1, 2, 4, and 6. Mock-treated reactions that did not include UV cross-linking but did include proteinase K (PK) were loaded in lanes 1 and 2, respectively. C, unlabeled competitor RNA titrates cross-linked proteins associated with exon 7. HeLa nuclear extract was incubated with no unlabeled RNA competitor (lane 2), NS competitor (lanes 3 and 4) or exon 7 competitor RNA (lanes 5 and 6) as indicated. A mock-treated reaction that did not include UV cross-linking was loaded as a control (lane 1). The protein size markers (kDa) are indicated to the left, and arrows denote sites of cross-linking bands of interest.