RON splicing and splicing complex formation <i>in</i><i>vitro</i>.

<p>(A) Diagrams of RON pre-mRNA substrates and their expected patterns of splicing. Boxes represent RON exons 10 (210 nt), 11 (147 nt) and 12 (166 nt), and lines represent introns 10 (87 nt) and 11 (80 nt) respectively. (B) Time courses of splicing in HeLa nuclear extract of the single intron substrates. Pre-mRNA substrates 10-11 and 10-11’ begin at the 5’ end of exon 10, with an additional GGG for transcription initiation, and end at the 3’ end of exon 11 plus a 7 nt intron portion of either wild-type exon 11 5’ splice site sequence (11,12) or consensus 5’ ss sequence (GUAAGUU) (10-11’). Pre-mRNA substrates 11-12 and 11-12’ begin at nt 7 of exon 11, which forms a natural GGG tract, and end either at the end of exon 12 (11,12) plus 7 nt of the wild type 5’ splice site or with an additional 3’ consensus 5’ss (11-12’). βG pre-mRNA is derived from β-globin exons 2 (226 nt) and part of exon 3 (56 nt), with a reduced intron (106 nt). DNA marker lengths are as shown (nt). (C) Analysis by native gel electrophoresis of pre-mRNA-containing complexes following incubation under conditions permitting assembly of complex E for the times shown. Nuclear extract was pre-incubated where indicated (U1 KD) with a 2’-O-methyl oligonucleotide complementary to the 5’ end of U1 snRNA prior to addition of pre-mRNA or incubated with heparin following assembly. Spliceosomal complexes E and H are labeled to the left of the panel. (D) Native gel electrophoresis after incubation of pre-mRNA in nuclear extract under conditions permitting spliceosome assembly. Complex H and the spliceosomal complexes A, B, C are labeled to the left of the panel.