Fig. 1: Prp28 contacts key proteins at the heart of spliceosome.
From: Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling
a Low ATP traps a transient interaction of Prp28 with the spliceosome. Splicing reactions (lanes 1–5) were done in V5-tagged Prp28 extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation without antibody (PAS; lanes 6–10) or with anti-V5 antibody (lanes 11–15). Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–15). Positions of pre-mRNA, splicing intermediates, and mRNA are indicated to the left. The experiment was repeated three times with similar results. b Schematic diagram showing a BPA-marked Prp28 cross-linked to protein X in a spliceosome assembled on MS2 stem-loop-tagged ACT1 pre-mRNA, which can be pulled down by MS2-maltose-binding protein-(MS2-MBP)-conjugated agarose beads. Thunderbolt, 365-nm UV irradiation. c Prp28-BPA cross-linked species (Prp28-X) detected by using anti-Prp28, or using anti-HA and anti-V5 tag antibody for Prp28-tagged experiments. K27, K41, K82, and K136 are the amino-acid residues in Prp28 replaced by BPA. (−) and (+), without or with UV irradiation, respectively. Filled circle, uncrosslinked Prp28. Asterisk, nonspecific background band. Detection of MS2-MBP serves as a loading control. The experiments were repeated three times with similar results. d Identification of the X proteins as Prp8, Brr2, Snu114, and U1C by using anti-Prp8, anti-Brr2, anti-Snu114, or anti-V5 (U1C-V5) antibody, respectively. The experiments were repeated three times with similar results. e Schematic summary of the cross-linking data. Splicing complexes accumulated at various ATP concentrations are shown to the left. The changing amount of Prp28 associated with the spliceosome is depicted to the right. Source data are provided as a Source Data file.
