RNA-editing-mediated exon evolution

Abstract

Background: Alu retroelements are specific to primates and abundant in the human genome. Through mutations that create functional splice sites within intronic Alus, these elements can become new exons in a process denoted exonization. It was recently shown that Alu elements are also heavily changed by RNA editing in the human genome.

Results: Here we show that the human nuclear prelamin A recognition factor contains a primate-specific Alu-exon that exclusively depends on RNA editing for its exonization. We demonstrate that RNA editing regulates the exonization in a tissue-dependent manner, through both the creation of a functional AG 3' splice site, and alteration of functional exonic splicing enhancers within the exon. Furthermore, a premature stop codon within the Alu-exon is eliminated by an exceptionally efficient RNA editing event. The sequence surrounding this editing site is important not only for editing of that site but also for editing in other neighboring sites as well.

Conclusion: Our results show that the abundant RNA editing of Alu sequences can be recruited as a mechanism supporting the birth of new exons in the human genome.

Figure 1

The birth of an Alu-exon through RNA editing. Editing prediction was inferred from alignment of cDNAs to human genomic DNA. (a) Schematic illustration of exons 7 to 9 of the NARF gene. Exons are depicted as blue boxes; the Alu-exon, derived from AluSx (AEx; purple box), is in a sense orientation and is shown in the middle. The intronic, antisense-orientation Alu sequence (AluS) is 25 base-pairs upstream of the exonized Alu. Sense and antisense Alus are expected to create a dsRNA secondary structure, thus allowing RNA editing. RNA editing changes an AA dinucleotide into a functional AG 3' splice site (lower panel). RNA editing also occurs in five positions in the Alu-exon itself (E1, E2, E3, E4 and E5). In the first position (E1), editing changes a UAG stop codon into a UGG Trp codon. (b) Predicted folding between the sense and antisense Alu sequences (upper and lower lines, respectively). Adenosines that undergo editing are marked by red. Splice sites utilized for Alu exonization are marked as 5' ss and 3' ss on the alignment.

Figure 2

Levels of Alu-exon inclusion and RNA editing in the endogenous human NARF gene. (a) cDNAs from various normal human tissues or cDNAs from various cell-lines were PCR amplified using primers specific for the two exons flanking the exonized Alu (upper and lower panels, respectively). The inclusion level of the Alu-exon is indicated at the top of the panel, and represents the total percentage of the Alu-containing mRNA isoform, where 100% corresponds to the total of both mRNA isoforms (inferred by the ImageJ program). Each PCR product was confirmed by sequencing. Schemata of the two mRNA products are shown on the right. (b) Editing efficiency in the five exonic sites (E1, E2, E3, E4 and E5; see Figure 1 for site positions) in different tissues and cell lines. The editing frequencies in each of the five edited sites, derived from sequence results obtained from an average of three independent amplifications, were quantified using the Discovery Studio Gene 1.5 program.

Figure 3

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.

Figure 4

Editing is directed by a specific sequence surrounding the editing nucleotide. (a) An illustration showing the positions that were mutated in the Alu-exon (AEx) and the antisense intronic AluS (AluS): the flanking nucleotides of the edited E1 site, and the position in the antisense Alu that is predicted to be opposite to the E1 in the dsRNA formation. (b) Chromas sequences of the Alu-exon editing of the wild-type (WT) and three mutants from (a). WT and mutant plasmids were introduced into 293T cells by transfection, total RNA was extracted, and splicing products were separated on 1.5% agarose gel following RT-PCR analysis. The Alu-exon inclusion of the WT and mutants is highly similar (not shown). The edited positions are highlighted in black. (c) Rounded editing frequencies of each of the five edited sites, from three separate experiments, were quantified using the Discovery Studio Gene 1.5 program.

Figure 5

The effect of editing in exonic sites on exon inclusion levels. Lane 1 represents a deletion of the Alu antisense and also a mutation that creates an AG at the 3'ss. This plasmid was used to generate an A-to-G mutation in each of the exonic edited sites (lanes 2-6). This is a similar analysis to that shown in Figure 3.