Spliceosome Assembly and Manipulation

U6 was depleted from Saccharomyces cerevisiae splicing extracts and splicing activity reconstituted with synthetic U6 snRNA, essentially as described^{28 (link)}. Spliceosomes were assembled on modified model pre-mRNA substrates: UBC4, for experiments probing branching, or ACT1, for experiments probing exon ligation, both synthesized by splint-mediated ligation^{49 (link)}. Oligonucleotides containing specific 5′ or 3′ splice site modifications were synthesized in house, as described previously^{50 (link)}. Assembled spliceosomes were isolated by affinity purification with Prp19p (ref. ^{36 (link)}), washed to remove ATP, and chased as described^{28 (link)}, at room temperature, in the absence of ATP, at pH 7.0, 8.0, or 8.5. All experiments were repeated with at least two independent extract preparations. Data were quantified using ImageQuant TL (Amersham Biosciences).