Alu-containing exons are alternatively spliced

Abstract

Alu repetitive elements are found in approximately 1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of the Alu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon. Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain an Alu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.

Figure 1

Schematic representation of the multiple alignment of the mRNAs of a microsomal glutathione transferase homolog gene with the genomic sequence. Three GenBank mRNAs (blue) align to the same genomic locus on chromosome 9, NT_008541 (red). Three ESTs that map to this locus are presented (purple), 38 other ESTs that align to the locus are not displayed to save space. Gaps in the alignment of mRNAs represent introns in the DNA. Four exons (marked I, II, III, and IV) are inferred from the presented alignment. Exon II is an alternative internal exon, contained entirely within an Alu repeat. Exon III is a constitutive internal exon, found in all detected splice variants and supported by seven expressed sequences (only five are shown). The LEADS output was searched for internal exons. A total of 1176 alternatively spliced internal exons were found, 61 of them (5.2%) contained an Alu fragment. A total of 4151 constitutive internal exons were found; none of them contained an Alu fragment.

Figure 2

Location of alternatively spliced internal exons within the mRNA. Data for 54 Alu-containing exons, for which there was noncontradictory information in the GenBank annotation, is presented in lighter shaded bars. Data of 62 alternatively spliced internal exons from chromosome 22, compiled by Hide et al (2001) are presented as reference (darker shaded bars).

Figure 3

Effect of exon insertion on the protein-coding region. Data for 45 Alu-containing exons occurring within the protein-coding region are presented in lighter shaded bars. Data of 48 alternatively spliced internal exons from chromosome 22 (Hide et al. 2001), which occur in the protein-coding region, are presented as reference (darker shaded bars). Exons were considered as domain adding if their length was a multiple of three, and there was no in-frame stop codon within them. Exons were considered as causing a premature termination either when they caused a frame-shift or when they presented an in-frame stop codon. Data for alternatively spliced internal exons from chromosome 22 were calculated from Table 2 in Hide et al. (2001).

Figure 4

Retention ratios of highly covered Alu-containing exons. Retention ratio for each exon was calculated by the number of expressed sequences that contain the exon as well as both flanking exons, divided by the total number of sequences that contain both flanking exons. Only the 31 exons with 10 or more total sequences that contain both flanking exons were taken for this analysis. Therefore, every exon represents ∼3% of the exons dataset.