Why are Alu exons alternatively spliced? (A) The splicing pattern of exonization events originating from the right arm of Alu elements (ExonRA) is alternative due to the presence of a downstream arm that presumably acts as a pseudo-exon. Thus, competition between two highly similar arms shifts the splicing pattern of the stronger arm to alternative. Although the weaker left arm does not undergo exonization, it nonetheless participates in the regulation of the stronger right arm by reducing the affinity of the splicing machinery for the exonized arm, thereby causing it to be alternatively spliced. (B) However, when the exonized right arm (ExonRA) is free of left arm regulation it is constitutively spliced. Such events are presumably deleterious, and therefore may be selected against.

Characteristics of Alu right arm exons and their counterpart intronic left arms. (A) Alignment of the right and left arm of AluJ consensus sequence (gi551536) in its antisense orientation (relative to the mRNA) using the MAVID alignment server (73). The PPT of the right arm was extended to 19 nt as, on average, the PPT length in exonized Alus is 19 bases ±3 (28) and is marked by horizontal brackets. The major 3′ss and 5′ss that are selected by Alu exons are indicated by arrows. Identical sequences are highlighted in gray. The 31-nt sequence that is present only in the right arm is indicated in a box. (B) Comparison of splice signals in the right and the left arms of 330 Alus that have undergone exonization originating from the right arm in the antisense orientation. Boxes indicate right and left arms. Locations of the real and putative splice sites are marked by arrowheads. Distribution of ESEs, based on ESEfinder, throughout the exon and the putative splice sites in the left arm are shown by a histogram divided into four bins, each of which represents a quartile of the exon length. The number in the center of the boxes represents the mean ESE density score based on ESEfinder. (C) Comparison of ESR densities between the right and left Alu arms. ESR densities are shown for three ESR datasets: ESEfinder (38), Goren et al. (39) and Fairbrother et al. (40).

The intronic left arm of the Alu element is essential for the alternative splicing of an upstream exon. (A) The sequence of an Alu retroelement in its antisense orientation from which exon 8 of ADAR2 is exonized. The right arm (in red) undergoes exonization while the left arm (in blue) is intronic. The potential PPT, 3′ss, and 5′ss in the left arm (unexonized) are underlined. ESE densities (according to ESEfinder) for each arm are indicated on the right. (B) Plasmids containing the indicated mutants were transfected into 293T cells. Total cytoplasmic RNA was extracted, and splicing products were separated in 2% agarose gel after reverse transcription polymerase chain reaction (RT–PCR). Lane 1, vector only (pEGFP); lane 2, splicing products of wild-type ADAR2; and lanes 3–10, splicing products of ADAR2 minigene mutated at the left arm of the Alu element in different combinations. ΔLA: deletion of the entire intronic left arm; ΔpPPT: deletion of the 14-bp pPPT sequence of the left arm; Δp3′ss: elimination of two potential 3′ss in the intronic left arm (AGAG to ACCG); Δp5′ss: elimination of two potential 5′ss in the intronic left arm (GTGT to GAAT). The two possible minigene mRNA isoforms are shown on the right. The numbers above the lanes indicate exon 8 inclusion level. (C) Insertion of an intronic left arm of an Alu element downstream to a constitutive exon results in a shift toward alternative splicing. The sequence of the intronic left arm of the Alu element, from which exon 8 of ADAR2 gene undergoes exonization (in its antisense orientation), was cloned downstream to constitutive exon 12 in the IMP minigene. The splicing assay was performed as described above. Lane 1, splicing product of wild-type IMP; lanes 2–3, splicing products of IMP minigene in which the intronic left arm of the Alu element from ADAR2 was inserted downstream to exon 12 in antisense and sense orientations, respectively; lanes 4–5, splicing products of IMP minigene in which the intronic left arm of the Alu element from ADAR2 was inserted upstream to exon 12 in antisense and sense orientations, respectively. The two possible minigene mRNA isoforms are shown on the right. The numbers above the lanes indicate exon 12 inclusion levels. (D): Exonization of the intronic left arm. Splicing assays were performed as described above. Lane 1, splicing products of wild-type ADAR2; lane 2, splicing products of ADAR2 mutant harboring a deletion of the intronic left arm; lane 3, splicing products of ADAR2 mutant harboring a deletion of the exonized right arm; lane 4, splicing products of ADAR2 mutant after strengthening of the p5′ss and pPPT of the intronic left arm; lane 5, splicing products of ADAR2 mutant harboring a replacement of the intronic left arm with the sequence of an additional right arm. The two minigene mRNA isoforms are shown on the right and were confirmed by sequencing.

Increasing the distance between the left and right arms of the Alu element results in a shift toward constitutive splicing. (A) Illustration of the Alu element in its antisense orientation. The right and left arms are marked by horizontal brackets. An intronic sequence of 350 bp was inserted in four different positions along the intronic Alu's left arm: (a) between the exonized right arm and the intronic left arm; (b) between the pPPT and the p3′ss of the intronic left arm; (c) in the center of the intronic left arm; (d) downstream of the intronic left arm. (B) Splicing assays were performed as described in Figure 2. Lane 1, splicing products of wild-type ADAR2; lanes 2–5, splicing products of ADAR2 minigene mutated as indicated in A. (C) Intronic sequences of growing lengths (10–300 bp) were inserted between the exonized right arm and the intronic left arm. Lane 1, splicing products of wild-type ADAR2; lanes 2–11, splicing products of ADAR2 minigene mutated as indicated.

The effect of pseudo splice signals and putative SR-binding sites within the left arm on the splicing pattern of the right arm. (A) Segments along the left arm 25 bp in length (rep1–rep5) were replaced with a designed sequence that did not contain any potential splice signals or SR-binding sites. The examined putative ESRs are shown. (B) Splicing assays were performed as described in Figure 2. Lane 2, splicing products of wild-type ADAR2; lanes 2–6, splicing products of ADAR2 minigene with replaced segments (rep1–rep5). (C) Lane 1, splicing products of wild-type ADAR2; lanes 2–6, splicing products of ADAR2 minigene mutated at the indicated binding sites in segments 2, with and without deletion of pPPT. (D) Changes in the binding capacities of putative SR proteins in segment 5 (near the potential 5′ss of the intronic left arm). Lane 1, splicing products of wild-type ADAR2; lanes 2–8, splicing products of ADAR2 minigene with the indicated mutations (see Supplementary Data).