Diverse splicing patterns of exonized Alu elements in human tissues

Abstract

Exonization of Alu elements is a major mechanism for birth of new exons in primate genomes. Prior analyses of expressed sequence tags show that almost all Alu-derived exons are alternatively spliced, and the vast majority of these exons have low transcript inclusion levels. In this work, we provide genomic and experimental evidence for diverse splicing patterns of exonized Alu elements in human tissues. Using Exon array data of 330 Alu-derived exons in 11 human tissues and detailed RT-PCR analyses of 38 exons, we show that some Alu-derived exons are constitutively spliced in a broad range of human tissues, and some display strong tissue-specific switch in their transcript inclusion levels. Most of such exons are derived from ancient Alu elements in the genome. In SEPN1, mutations of which are linked to a form of congenital muscular dystrophy, the muscle-specific inclusion of an Alu-derived exon may be important for regulating SEPN1 activity in muscle. Realtime qPCR analysis of this SEPN1 exon in macaque and chimpanzee tissues indicates human-specific increase in its transcript inclusion level and muscle specificity after the divergence of humans and chimpanzees. Our results imply that some Alu exonization events may have acquired adaptive benefits during the evolution of primate transcriptomes.

Figure 1. Examples of “Correlated” Exons analyzed by Exon Array analysis, semi-quantitative RT-PCR and sequencing.

A. Exon array analysis of NLRP1. B. RT-PCR analysis of Alu-derived exon in NLRP1. C. Exon array analysis of FAM55C. D. RT-PCR analysis of Alu-derived exon in FAM55C. E. RT-PCR analysis of Alu-derived exon in SLFN11. F. RT-PCR analysis of Alu-derived exon in NOX5. In Exon Array analysis, the bold line represents the overall gene expression levels across all 11 tissues, each with 3 replicates; each of the fine lines represents the background corrected intensities of a probe targeting the Alu-derived exon. The Pearson correlation coefficient of the individual probe's intensities with the estimated gene expression levels in 11 tissues is shown at the top right corner of each graph. In each gel figure, solid arrows show sequencing analysis confirmed Alu exon inclusion forms. Hollow arrows show sequencing analysis confirmed Alu exon skipping forms.

Figure 2. Examples of tissue-specific Alu-derived exons analyzed by Exon Array analysis, semi-quantitative RT-PCR and sequencing.

A. Exon array analysis of ICA1 indicates a testes specific inclusion of Alu-derived exon. B. RT-PCR analysis of Alu-derived exon in ICA1. C. Exon array analysis of ZNF254 indicates a cerebellum specific inclusion of Alu-derived exon. D. RT-PCR analysis of Alu-derived exon in ZNF254. E. RT-PCR analysis of Alu-derived exon in PKP2. F. RT-PCR analysis of Alu-derived exon in SEPN1. In Exon Array analysis, the bold line represents the overall gene expression levels across all 11 tissues, each with 3 replicates; each of the fine lines represents the background corrected intensities of a probe targeting the Alu-derived exon. In each gel figure, solid arrows show sequencing analysis confirmed Alu exon inclusion forms. Hollow arrows show sequencing analysis confirmed Alu exon skipping forms. Dashed arrows show sequencing analysis confirmed non-specific PCR products.

Figure 3. Evolution of SEPN1 Alu-exon splicing in primates.

A. The splicing pattern of SEPN1 Alu-derived exon. B. RT-PCR analysis of the SEPN1 Alu-derived exon in human, chimpanzee and macaque tissues. The RT-PCR primer was designed from the upstream and downstream constitutive exon on the human gene and matched perfectly to chimpanzee and macaque transcripts. C. Realtime qPCR primers that specifically amplify exon inclusion and skipping forms. The reverse PCR primer for the skipping form was designed from the junction of upstream and downstream constitutive exons. These PCR primers perfectly matched both human and chimpanzee transcripts. D. The ratio of exon inclusion/skipping in human tissues and tissues of two chimpanzees estimated by realtime qPCR. The SEPN1 exon showed strong exon inclusion in human muscle but not in chimpanzee muscle. C, cerebellum; K, kidney; L, liver; M, muscle.

Figure 4. Most Alu exons with substantial transcript inclusion levels are derived from ancient Alu elements in the human genome.

Plotted here are distributions of AluJ Class and AluS Class in the human genome, in Alu-derived internal exons, and in Alu-derived exons with substantial transcript inclusion levels based on our RT-PCR results. AluJ class is indicated by white column; AluS class is indicated by hatched column.