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Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer - PubMed

  • ️Sat Jan 01 2005

Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer

Haixin Lei et al. Nucleic Acids Res. 2005.

Abstract

Ancient Alu elements have been shown to be included in mature transcripts by point mutations that improve their 5' or 3' splice sites. We have examined requirements for exonization of a younger, disease-associated AluYa5 in intron 16 of the human ACE gene. A single G>C transversion in position -3 of the new Alu exon was insufficient for Alu exonization and a significant inclusion in mRNA was only observed when improving several potential splice donor sites in the presence of 3' CAG. Since complete Alu exonization was not achieved by optimizing traditional splicing signals, including the branch site, we tested whether auxiliary elements in AluYa5 were required for constitutive inclusion. Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 222-228 and point mutations in highly conserved nucleotides of this heptamer decreased Alu inclusion. In addition, we show that Alu exonization was facilitated by a subset of serine/arginine-rich (SR) proteins through activation of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE minigenes generated similar splicing patterns in both ACE-expressing and non-expressing cells, suggesting that previously reported allelic association with plasma ACE activity and cardiovascular disease is not attributable to differential splicing of introns 16 and 17.

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Figures

Figure 1
Figure 1

A lack of a haplotype-specific pre-mRNA splicing pattern of ACE minigenes. (A) Construction of ACE minigenes. Minigenes A and B were amplified with primers 1 and 2 and a DNA sample with defined AluYa5 I/D genotype. Minigenes C and D were prepared by deleting a segment of intron 16 with a combination of vector primers and primers 3 and 4. Minigenes E and F were constructed by deleting tandemly arranged sense Alu repeats AluSx and AluJb using primers 4 and 5. Antisense AluYa5 was present in constructs A, C and E. Primers are shown as black arrows. Exons (E) are represented by boxes, introns by lines. The position and orientation of Alu sequences is denoted by grey arrows. Exons, introns and Alus are shown to scale; scale units are kilobases (kb). Exon and intron numbering is consensual as referred to in previous studies (52,54). Location of three tested SNPs around the splice acceptor site of intron 16 is shown near the 3′ss of intron 16 (the upper panel). Putative susceptibility (minigenes B, D and F) and protective (A, C and E) haplotypes in the indicated SNP are shown as 3 nt above each construct (the lower panel). Lines in the lower panel represent DNA sequences of the ACE gene that are present in the indicated minigene, whereas deleted sequences are highlighted with brackets. Alu-E (for Exonized Alu) and Alu-L (for a Long isoform retaining intron 17 and 3′ end of intron 16) represent RNA products generated by mutated constructs (see Figures 2–4). Alu-E isoforms spliced to the predicted 5′ss (black arrow) and positions −16 and +136 relative to the predicted 5′ss are shown below the I/D AluYa5. (B) Haplotype-specific minigenes A–F were transfected into 293T cells and their splicing pattern was examined by RT–PCR 48 h post-transfection. Exon 17 skipping was not observed after 50 PCR cycles (data not shown). (C) A splicing pattern of a truncated minigene individually mutated in the indicated SNPs. We mutated minigene B, which has unabridged intron 16 and lacks AluYa5 to study a putative effect of these mutations in the natural context and to facilitate mutagenesis.

Figure 2
Figure 2

Alu exonization generated by minigenes mutated in BPS and 3′ and 5′ss. Alu inclusion in mRNA was tested following an introduction of a series of mutations at the 3′ss, 5′ss and the BPS in a minigene transfected into 293T cells. (A) Inclusion of AluYa5 in mature transcripts following mutations in the 5′ss, 3′ss and the BPS in minigene E, which lacks most of intron 16 to simplify overlap-extension PCR and reduce the proportion of constructs with undesired mutations. AI, Alu inclusion levels as a mean (and SD) of triplicate transfections into 293T cells. Inclusion of AluYa5 in mRNA is indicated by a light grey rectangle on the right side. WT, wild-type BPS CAGUCA−19C. M, mutation in putative BP A−19>T; Y, yeast (S.cerevisiae) BPS UACUAA−19C. Adenosine −19 was also predicted as a putative BP (BPS score 3.25) through comparison of human and mouse introns (

). (B) Nucleotide sequence of the junction (arrow) between upstream exon and exonized Alu; (C–E) boundaries (arrow) between the exonized Alu and downstream exon generated by optimizing the 5′ss in position −16 (C; see lanes 3 and 4 in Figure 2A), in the predicted splice sites (D; lanes 7, 10, 11 and 13 in Figure 2A) and in position +136 (E).
Figure 3
Figure 3

Identification of a splicing enhancer element in ACE intron 16 AluYa5 Influence of segments 2–5 deletions and segment 2 point mutations on Alu exonization. (A) Exonized Alu segment (upper case) with putative ESEs (underlined and numbered 1–6). Segments 1, 2 and 6 were predicted by the ESE Finder (8), segments 1 (C allele only), 3 and 4 were significant ESEs identified through octamer sequences (9) and segment 5 was predicted by the RESCUE-ESE (7). Intronic sequence is in lower case. To facilitate mutagenesis, segments 2 through 5 were individually deleted in minigene E′. This minigene contained intron 16 truncation (Figure 1A) and four point mutations optimizing the splice sites (shown below the nucleotide sequence in bold). A segment 2, which was mutated individually in each position, is in the middle of the sequence in bold. (B) Alu inclusion levels following deletions of segments 2–5 and point mutations in segment 2. Transfection of all splicing reporters was into 293T cells. AI, Alu inclusion. (C) Alu inclusion levels in double mutants of minigene E. (D) Consensus sequence of segment 2 heptamer in alternatively spliced exons containing right arms of antisense Alus as identified previously (34). Representation of each heptamer position (numbered above) and flanking sequences was visualized using a pictogram utility available at

.
Figure 4
Figure 4

SR-mediated activation of the 3′ss of ACE AluYa5. Minigenes A and E were co-transfected with plasmids expressing a subset of SR proteins. (A) Inclusion of ACE IVS16 AluYa5 in mature transcripts in 293T cells transfected with 0.5 μg of minigene A containing 3′ CAG and 1 μg of plasmids expressing the indicated proteins. (B) RRMs are critical ASF/SF2 domains for the Alu-L formation. ASF-dRRM1, ASF-dRRM2, ASF-dRS and ASF-dGly denote ASF/SF2 lacking RRM1, RRM2, the RS domain and a heptaglycine stretch, respectively. WT, wild-type. Inclusion levels are mean values (SD) of two independent transfection experiments.

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