Addition of the 5′ss oligo to the affinity-purified 37S exon complex induces a shift to a 45S complex. (A) Glycerol-gradient centrifugation (75 mM KCl) of affinity-purified 45S B-like complexes or 37S exon complexes incubated alone or solely with the indicated 5′ss oligonucleotide. The percentage of total radioactivity is plotted for each gradient fraction. (B) RNA composition of complexes from peak gradient fractions 14–16 (37S exon or + 2′Ome 5′ss oligo) or fractions 16–18 (+5′ss oligo or 45S B-like). RNA was separated by denaturing PAGE and visualized by silver staining. Positions of the snRNAs and MINX exon RNA are indicated. (C) Western blot of the indicated complexes ± the 5′ss oligo immunostained with antibodies against the phosphorylated form of hPrp31 (hPrp31 phos). Antibodies against hSnu66 were used to ensure equal loading.
