(A) Diagram of exon complex purification. MS2-tagged WT and MUT N1 exons were incubated with the MS2-MBP fusion protein prior to assembling of exon complexes. The reactions were resolved on glycerol density gradients, peak fractions were pooled, and complexes were affinity purified on amylose beads. Note that the MUT RNA also forms larger complexes found at the bottom of the gradient. (B) Top panel: RNA was extracted from exon complexes purified as in (A), resolved on 8% urea-PAGE, and stained with SYBR Gold. Total nuclear RNA is shown to the left with each U snRNA indicated. The asterisk (*) marks uncharacterized band. Bottom panel: Intensities of the N1 exon, U1 and U2 bands normalized by their nucleotide length are plotted relative to the N1 exon for each complex. (C) MS2-tagged WT and MUT N1 exon RNAs were assembled into exon complexes in the absence of ATP to prevent progression to the EDC, purified on amylose beads, washed in increasing concentrations of potassium glutamate (80, 100 or 150 mM) and eluted in maltose. Proteins in each complex were resolved on SDS-PAGE and immunoblotted with antibodies against PTBP1, U2AF65 and CBP80 (loading control). (D) Diagram of the 3’ splice site, labeled at the AG (red asterisk). Labeled WT and MUT RNAs were assembled in splicing reactions for 10 min before irradiation in 254-nm UV to crosslink proteins to the RNAs. Reactions were treated with RNAse T1, denatured in 0.1% w/v SDS at 95°C, immuno-precipitated with α-U2AF65 antibody or mouse IgG (control), resolved on SDS-PAGE, and autoradiographed. The band of crosslinked U2AF65 is indicated.