Mapping BP of exon 3. (A) In vitro splicing of construct containing the HTR4 exon 3 AGEZ. Splicing precursor, intermediates, and products are depicted to the right of the gel, with the α-Tropomyosin exons TM2 and TM3 in boxes, the AGEZ in gray, and introns in black. Looped RNA-forms correspond to the lariat and lariat intermediates. Lane con, unspliced control; sp, spliced (2-h time point); db, spliced and then debranched. (B) Primer extension map BP to position –151. Left panel shows sequencing lanes, right panel primer extension with control RNA in the splicing buffer (con), spliced transcripts (sp), and RNA that was debranched after splicing (db). Sequence surrounding the BP is depicted on the left side of the gel. (C) Mutagenesis of BP in exon trapping construct: lane 1 shows WT splicing pattern with mainly exon inclusion. Upon mutation (lanes 2–4) of more As into Gs (sequence changes in bold in box beside the gel) splicing pattern changes to exon skipping and usage of alternative 3′ ss (exon incl I– III). Percentage of exon inclusion (n = 3) ± standard deviation is shown underneath the gel. Control lanes are reverse transription reactions without an enzyme (−RT) and PCRs without a template (0). (D) The sequence of exon 3 and its AGEZ: intron sequence in lower case and the exon sequence in upper case; first upstream AG dinucleotide and 3′ ss AG in bold. Positions of BP are underlined and BP-A is in upper case. Alternative 3′ ss highlighted in boxes with stars correspond to the three different alternative 3′ ss products in C).
