FIG. 3.

5′ss selection in Alu exons. (A) Diagram of the ADAR2 minigene containing exons 7, 8, and 9. Exon 8 is an Alu-derived exon; the three shades of gray represent the three potential 5′ss regions A, B, and C. The last three exonic and first six intronic nucleotides of the wt and mutated 5′ss are shown, marked as A to A2, B0 to B3, and C to C2. Senapathy scores of the 5′ss are given in parentheses. (B) Plasmids containing the indicated mutants were introduced into 293T cells by transfection. Total cytoplasmic RNA was extracted, and splicing products were separated in a 2% agarose gel after RT-PCR. Lane 1, splicing products of wt ADAR2 (marked B1 in panel A); lanes 2 to 11, splicing products of the indicated mutants. The numbers above the lanes indicate the percentages of inclusion of exon 8. The three minigene mRNA products are shown on the right. Empty boxes define constitutive exons, and the shaded boxes indicate the selection of either of the three 5′ss. (C) Diagram showing the PGT minigene, containing exons 9, 10, and 11. Exon 10 does not exonize under endogenous conditions but constitutes an intronic Alu element on the verge of exonization. The three shades of gray in exon 10 represent three potential 5′ss (sites A, B, and C). Exons 9 and 11, shown by empty boxes, are constitutively spliced. The wt 5′ss (marked A1) and six mutated 5′ss are shown in the lower part, marked as A2, B to B3, and C to C2. Senapathy scores of the 5′ss are presented as described above. (D) Analysis of in vivo splicing essentially as described for panel B. Lane 1, splicing products of wt PGT (marked A1 in panel C); lane 2, splicing products of a 3′ss mutant (from GAG to TAG); lanes 3 to 12, splicing products of the indicated mutants. The three minigene mRNA products are shown on the right. Empty boxes indicate constitutively spliced exons, whereas the boxes colored in three shades of gray indicate selection of 5′ss A, B, or C. All PCR products were verified by sequencing.