Comparative analysis of transposed element insertion within human and mouse genomes reveals Alu's unique role in shaping the human transcriptome

Abstract

Background: Transposed elements (TEs) have a substantial impact on mammalian evolution and are involved in numerous genetic diseases. We compared the impact of TEs on the human transcriptome and the mouse transcriptome.

Results: We compiled a dataset of all TEs in the human and mouse genomes, identifying 3,932,058 and 3,122,416 TEs, respectively. We than extracted TEs located within human and mouse genes and, surprisingly, we found that 60% of TEs in both human and mouse are located in intronic sequences, even though introns comprise only 24% of the human genome. All TE families in both human and mouse can exonize. TE families that are shared between human and mouse exhibit the same percentage of TE exonization in the two species, but the exonization level of Alu, a primate-specific retroelement, is significantly greater than that of other TEs within the human genome, leading to a higher level of TE exonization in human than in mouse (1,824 exons compared with 506 exons, respectively). We detected a primate-specific mechanism for intron gain, in which Alu insertion into an exon creates a new intron located in the 3' untranslated region (termed 'intronization'). Finally, the insertion of TEs into the first and last exons of a gene is more frequent in human than in mouse, leading to longer exons in human.

Conclusion: Our findings reveal many effects of TEs on these two transcriptomes. These effects are substantially greater in human than in mouse, which is due to the presence of Alu elements in human.

Figure 1

How TEs affect the human and mouse transcriptome. (a) Summary of the effect of (i) exonization of TEs on the transcriptome; of the effect of exonization that (ii) creates an alternatively skipped exon, (iii) transforms an existing exon to an alternative 5'ss exon, or (vi) transforms an existing exon to an alternative 3'ss exon; or of the effect of exonization that (v) creates a constitutively spliced exon. The table on the right shows the corresponding numbers of transposed elements (TEs). (b) Summary of the effect of TE insertions in the first or last exon. Panel i shows the insertion of TEs (gray box) into an exon (white box). The insertion of the TEs can cause an enlargement of the first or last exon (panels ii and iii), or, in some cases, activates intronization (generating an alternatively spliced intron that splits the last exon into two smaller exons; panel iv). The numbers of those events according to TE family are shown on the right-hand side.

Figure 2

RT-PCR analysis of selected Alu and MIR exons. (a) Multiple alignment of mammalian interspersed repeat (MIR) exon in DMWD gene among mammals. Exon sequences are marked in blue, flanking intronic sequences are marked in black, and the canonical AG and GT dinucleotides at the 3'ss and 5'ss are marked in red. Nucleotide conservation is marked at the lower edge, with asterisks indicate full conservation and colons indicating partial conservation relative to the MIR consensus sequence (lower row). The divergence in percentage from the consensus MIR sequence is indicated under (MIR div); exon conservation in percentage compared with the human exon is indicated under (exon conserve); EST/cDNA accession confirming the exon insertion is indicated under (cDNA/EST holding evidence), and skipping is indicated under (cDNA/EST skipping evidence). Nonconserved nucleotides are marked in yellow. (b) This panel is similar to panel a, except that the conservation is shown for the protein coding sequence. (c) Total RNA was collected from SH-SY5Y human cell line and mouse brain tissue. Reverse transcription polymerase chain reaction (RT-PCR) analysis amplified the endogenous mRNA molecules using primers specific to the flanking exons. The PCR products were separated on an agarose gel, extracted and sequenced. A schema of the mRNA products is shown on the left and right. Columns 1 to 4 show the splicing pattern of orthologous human (H) and mouse (M) exons originating from the MIR element. Columns 1 and 2 show alternative splicing of an ortholog MIR element in both human and mouse, respectively (exon 4 in DMWD gene), and columns 3 and 4 show a constitutive pattern in both species (exon 5 in the MYT1L gene). Column 5 shows constitutive splicing of an Alu element in the human exon 3 of FAM55C gene. All PCR products were confirmed by sequencing. We cannot fully reject the option that an exon that is constitutively spliced under the above conditions is alternatively spliced in other cells or conditions. However, the constitutive selection is also supported by EST/cDNA coverage.

Figure 3

Alu insertions into an exon activate intronization in the CWF19L1 gene. (a) Intronization. (i) Illustration of the last exon of the CWF19L1 gene in mouse. (ii) During primate evolution, two Alu elements were inserted into the exon. (iii) Because of these insertions, an intronization process activates two splice sites within the exon, a 3' and a 5' splice site. The isoform in which the intron is spliced out is supported by 12 mRNA/expressed sequence tags (ESTs), and the isoform in which the intron is retained is supported by four mRNA/ESTs. (b) Testing the splicing pathway of this exon between human and mouse. Polymerase chain reaction (PCR) analysis on normal cDNAs from human kidney (marked H) and from mouse brain tissue (marked M). PCR products were amplified using species-specific primers, and splicing products were separated in 1.5% agarose gel and sequenced. (c) Alignment of the sequence of the last exon of the CWF19L1 gene among human, mouse, rat, and dog is shown. The two Alu elements are marked in gray. The selected 5'ss and 3'ss are marked.

Figure 4

Characteristics of the exonized TEs. (a) The free energy resulting from the base pairing of the 5'ss with U1 snRNA (ΔG) for the indicated exonized transposed elements (TEs). 'const.' and 'alt.' indicate conserved human and mouse constitutively spliced and alternatively spliced exons, respectively. (b) The divergence in percentage from the consensus sequence. (c) The average inclusion level. (d) The average exon length.

Figure 5

5' and 3' splice site selection of Alu, B1, and MIR. Both Alu and B1 originated from 7SL RNA. Alu has a dimeric form with a very similar left and right arm, whereas B1 has a monomeric form similar to the left arm of the Alu element. (a) The most selected 5'ss and 3'ss within the exonized Alu element in the antisense orientation. The right and left arm are shown by boxes. The numbers within the boxes indicate positions (according to the Alu consensus sequence) in which the prevalent 3'ss and 5'ss are selected, above and below the boxes, respectively. Pictogram depiction of each splice site is shown, the consensus sequence of Alu is marked above the pictogram, and the number of times that the site was selected is shown below it. (b and c) Similar presentation as in panel a for the prevalent 5'ss and 3'ss selected in B1 and mammalian interspersed repeat (MIR) elements, respectively.