Intronic Alus Influence Alternative Splicing
Figure 3
Editing sites within the intronic Alus.
(A) Schematic illustration of exons 2 to 3 of the RABL5 gene. Exons are depicted as black boxes; the intronic Alus, derived from AluJo and AluSx, in sense and antisense orientations, respectively, are shown in the middle gray-shaped boxes. The intronic, antisense Alu sequence (AluSx) is 102 nucleotides downstream of the sense AluJo and AluSx is 24 nucleotides upstream of the junction of exon 3. Sense and antisense Alus are expected to form a double-stranded secondary structure, thus allowing RNA editing. (B) Editing sites were inferred from alignment of five cDNAs (accession numbers BC050531, BC038668, BI547904, BI548328, and DB495755) to the human genomic DNA. RNA editing occurs at eight positions within the antisense AluSx and at eleven positions within the sense AluJo. Based on these editing sites, the pairing between the sense and antisense Alu sequences was inferred (upper and lower lines, respectively). The region in which editing occurs starts at the middle of the right arm (position 232 in the AluSx consensus) and ends at the beginning of the left arm of Alu2 (position 101 in the AluSx consensus). Panel B shows only this corresponding region, while the entire Alu-Alu potential dsRNA is shown in Figure S3. (C) To further confirm the editing activity, total RNA was extracted from a neuroblastoma (SH-SY5Y) cell-line and treated with DNaseI, followed by RT-PCR analysis using primers to exon 2 and exon 3, and to intron 2 and exon 3 (lanes 1 and 2, respectively; see also Materials and Methods). The PCR products were sequenced. (D) The upper PCR product shown in panel C lane 2 was cloned and sequenced. The Chromas sequence is shown with the editing sites, found in AluSx, marked by boxes. (E) Editing in Alu2 requires the presence of Alu1. Wild type RABL5 minigene (WT) and a mutant in which Alu1 was deleted (ΔAluJo) were transfected into 293T cells. RNA was extracted, treated with DNase I, and amplified using set of primers flanking Alu2 and designed to amplify only exogenic transcripts. Sequencing chromatograms of four nucleotides in AluSx are shown (this editing site is also marked in green in panel B).