The antisense Alu is essential for exonization. (a) An illustration of the NARF minigene that was constructed, containing the genomic sequence of the human NARF gene from exon 7 to 9. The sites that were mutated in (b) are shown. (b) The minigene was transfected to human 293T cells, and total RNA was collected and examined by RT-PCR analysis using specific primers to mRNA products of the plasmid minigene. The first lane is the wild-type (WT) pattern. Lanes 2 and 3 represent a deletion of the antisense intronic Alu. Lane 3 also represents an AA→AG mutation at the 3'ss. Lanes 4 and 5 represent an AA→AT and AA→AG mutation at the 3'ss (without deletion of the antisense Alu), respectively. The inclusion level of the Alu-exon is indicated at the top of the gel, and represents the total percentage of the edited-Alu-containing mRNA isoform, where 100% corresponds to the total of both mRNA isoforms (inferred using the ImageJ program). Schemata of the two mRNA products are shown on the right.