SR proteins are required for the first step of the splicing reaction with RNA substrates containing 3, 2, or 1 exon nucleotide. (A) The SR protein 9G8 (200 nM) can complement S100 extract-dependent lariat formation of AdML RNA substrates containing 3, 2, or 1 exon nucleotide. (B) The SR proteins SC35 (500 nM), ASF/SF2 (200 nM), and a preparation of total SR proteins from nuclear extracts (100 ng/25 μl reaction) can also complement S100-dependent lariat formation of an AdML minimal substrate containing 3 exon-1 nucleotides. (C and D) The SR protein 9G8 (200 nM) complements S100 extracts to reconstitute both steps of splicing for exon-containing substrates and the first step of splicing for minimal substrates derived from two other pre-mRNAs, ftz (C) and β-globin (D).