Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

Displacement of Cwc24 from the spliceosome during Prp2-mediated catalytic activation, implying its noninvolvement in catalytic step 1 of splicing. (A) Affinity-purified B^actΔPrp2 complexes assembled on Atto647N-M3Act, carrying Cwc24-EGFP (column 1), were complemented as indicated above each bar, or (B) were eluted from the affinity column and incubated with increasing concentrations of KCl for 30 min on ice before dcFCCS measurements, as shown. (C) Western blot analysis of yeast splicing extracts carrying Cwc24 tagged with the TAP tag before and after depletion of Cwc24 (ΔCwc24). (D) A uniformly ³²P-labeled M3Act pre-mRNA was incubated in yeast whole-cell extract, which was either nondepleted (lanes 1–5) or Cwc24-depleted (lanes 6–20) under standard splicing conditions. Recombinant Cwc24 was then added to a final concentration of 1 or 3 μM. The splicing mixtures were incubated at 23°C and stopped at the time indicated. RNA was analyzed on an 8% urea–polyacrylamide gel and visualized by autoradiography. The positions of the pre-mRNAs, the splicing intermediates, and products are indicated on the right. The lariat-intron and the mRNA observed after 20-min incubation were quantified using Quantity One software (BioRad), and the results are shown below the gel. (E) Affinity-purified B^actΔPrp2 complexes assembled on ³²P-labeled M3Act, carrying Cwc24-EGFP (lane 1), were complemented with Prp2, Spp2, and ATP (lane 2), plus the addition of Cwc25 (lane 3). The positions of the pre-mRNAs, the splicing intermediates, and products are indicated on the left.