FIGURE 5.

Mutation of nucleotides at the exon–intron junction of the 5′ss oligo abolishes 45S B-like complex formation. (A) Schematic of interactions between the 5′ss oligonucleotide and U5 snRNA, U6 snRNA, and hPrp8, and overview of mutations introduced within the 5′ss oligonucleotide sequence to abolish base-pairing with U5 and/or U6 snRNA. Mutated nucleotides are highlighted in gray. (B) Native gel analysis of B-like complex assembly upon addition of the indicated 5′ss oligonucleotides to the splicing reaction after allowing cross-exon complex formation. Samples were analyzed on a 2% (w/v) LMP agarose gel in the presence of heparin and visualized by autoradiography. The identities of the spliceosomal complexes are indicated. (C) Glycerol-gradient centrifugation (150 mM KCl) of spliceosomal complexes formed on ³²P-MINX exon RNA in splicing reactions upon addition of the indicated 5′ss oligonucleotides. The percentage of total radioactivity is plotted for each gradient fraction.