FIG. 6.

The −34 silencer element blocks prespliceosome E′ complex formation. (A) Diagram of pre-mRNA substrates. Formation of E′ or E complex on different RNA substrates in the untreated extract (NE), U2AF⁶⁵-depleted extracts (ΔU2AF), or ΔU2AF extract supplemented with recombinant His-U2AF⁶⁵. The migration positions of the complexes are shown on the right. (B) HeLa extract. (C) CA77 extract. (D) Quantitative evaluation of the E′ or E complex formation shown in panel B. Ratio of the E′ to H or of E to H (amount of E′ or E complex divided by amount of the H complex) was calculated based on three independent experiments. (E) Quantitative evaluation of the E′ or E complex formation shown in the panel C.