Intronic Alus Influence Alternative Splicing (original) (raw)
Figure 3
Editing sites within the intronic _Alu_s.
(A) Schematic illustration of exons 2 to 3 of the RABL5 gene. Exons are depicted as black boxes; the intronic Alus, derived from _Alu_Jo and _Alu_Sx, in sense and antisense orientations, respectively, are shown in the middle gray-shaped boxes. The intronic, antisense Alu sequence (_Alu_Sx) is 102 nucleotides downstream of the sense _Alu_Jo and _Alu_Sx is 24 nucleotides upstream of the junction of exon 3. Sense and antisense _Alu_s 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 _Alu_Sx and at eleven positions within the sense _Alu_Jo. 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 _Alu_2 (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 _Alu_Sx, marked by boxes. (E) Editing in _Alu_2 requires the presence of _Alu_1. Wild type RABL5 minigene (WT) and a mutant in which _Alu_1 was deleted (Δ_Alu_Jo) were transfected into 293T cells. RNA was extracted, treated with DNase I, and amplified using set of primers flanking _Alu_2 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).