SVA and alternative splicing at the MAST2 locus. (A) The canonical SVA is displayed, consisting of some number of CCCTCT hexameric repeats followed by the Alu-like region, a variable number of tandem repeats, and then the SINE-R region, followed by a polyA signal with the entire SVA flanked by target-site duplications (black arrowheads). Two black arrows over the Alu-like region indicate sequence homology in the SVA to two ancestral Alu elements. (B) Shown is an ancestral CH1 MAST2 locus, sometime after the human–chimp split, containing a full-length SVA within intron 1 (top). An alternative splicing event into the Alu-like region (black line) likely occurred resulting in the original SVA_F1 founder insertion (bottom) flanked by target-site duplications (black arrowheads) in the human genome. Note the loss of the entire CCCTCT hexamer and most of the Alu-like region after the alternative splicing event. The SVA splice site relative to SVA_Rep is aligned to the known splice site sequence (middle).

Identification of the SVA master locus on CH10. (A) The SVA insertion on CH6. The 40-bp deletion in the MAST2 sequence (△) allowed the identification of the (B) CH6 progenitor element to be identified on CH3. The SVA insertion on CH3 contains both 5′ and 3′ tranductions. At the 5′ end, the SVA contains a truncated AluSc while at the 3′ end, an AluSp along with additional sequence, indicated by X, followed by a polyA tail, with the entire insertion flanked by target-site duplications. The 3′ transduction (X, red line) on CH3 allowed the identification of (C) the master element on CH10 along with 12 additional elements derived from the CH10 locus. The SVA on CH10 contains starting at it's 5′ end: 185 bp transduction derived from CH9, a full-length AluSc, the MAST2-SVA, an AluSp, polyA tail, and then a unique sequence, which was 3′ transduced, and which allowed the identification of CH10 as the source locus. A target-site duplication flanks the insertion, which inserted on CH10. Downstream polyA signals are displayed over the X and Y transduction sequences.

Genesis of the SVA master locus on CH10. The AluSc on CH9 and the AluSp on CH10, hereafter 5′ Alu and 3′ Alu, respectively, are shown present in the human–chimp common ancestor. The original target-site duplications (TSDs) for the 5′ Alu and the 3′ Alu are indicated by blue and red arrowheads, respectively. (A) After the human–chimp split, the initial SVA_F1 was formed due to an alternative splicing event. A SVA mRNA was transcribed from either the founder SVA_F1 insertion or some other unknown SVA_F1 locus in the human genome, derived from the original SVA_F1 founder insertion. (B) The SVA mRNA is retrotransposed directly downstream from the 5′ Alu on CH9 as displayed by the SVA insertion flanked by TSDs (green arrowheads). (C) Transcriptional initiation occurred upstream of the 5′ Alu, generating a composite mRNA containing in order (1) 185 nt of 5′ transduced sequence from CH9, (2) the 5′ Alu, and (3) the SVA_F1. (D) The composite mRNA from CH9 retrotransposed to CH10 directly upstream of the 3′ Alu. (E) The CH10 master element present in the human genome reference is shown. Directly downstream from the 3′ Alu TSD, unique sequence blocks (blue boxes), labeled X and Y, are present in the daughter insertions derived from this locus. The polyA signals utilized in the 3′ transductions are indicated by AATAAA relative to the unique sequence blocks.

SVAs contain many transcriptional start sites (TSSs). 5′ RACE was performed on total RNA extracted from 293T, HeLa, and nTera cell lines, along with chimp testes. A nested PCR was performed followed by Sanger sequencing of Topo clones. The 5′ RACE adaptor was identified, and the first nucleotide after it was annotated as the TSS. Human (red triangles) and chimp TSS (blue triangles) within SVA are aligned relative to the position in SVA_Rep. The CCCTCT hexamer (Hex), Alu-like region, and part of the VNTR are shown. TSSs aligning upstream of SVA are displayed relative to the first nucleotide of the SVA insertion in the genome. Both human (red triangles) and chimp (blue triangles) TSSs identified by 5′ RACE are shown.

SVA gene-trapping has occurred in other hominids. (A,B) Two different SVA splicing events followed by retrotransposition derived from the TPTE locus on chromosome 21 flanked by a target-site duplication. (A) CH3 SVA containing four processed TPTE exons that utilized AG 336. (B) CH15 SVA containing five processed exons that utilized AG 386. (C) The structure of SVA insertions on CH13, CH18, and CH21 containing the six processed exons from the RHOT1 gene on CH17 with identical target-site duplications.

SVA gene-trapping and exonization are not rare. (A) Two SVAs were cloned into PKC-EGFP () to test the mutagenic potential of SVA splicing. Primers used for RT-PCR are marked. (B) RT-PCR was performed on total RNA extracted from 293T cells transiently transfected with pPKC-EGFP containing one of two different SVAs cloned into the intron. SVA exonization events (left panel) are annotated with the first and last nucleotide of the SVA exon, all of which occur within the Alu-like and VNTR domains. A representative agarose gel displaying SVA alternative splicing events is shown (right panel) (see ). (*) Indicates bands verified by DNA sequencing to be SVA splicing events. (C) Semi-quantitative PCR to determine the frequency of SVA exonization. Ten cycles of PCR on cDNA from individual pPKC-EGFP, pPKC-SVA_C2CD3, and pPKC-SVA_MTFR1 transfections were carried out using PKC For and 1R. PCR products were resolved on a 2% agarose gel, followed by overnight transfer to a membrane, and subsequent probing using a DNA probe targeting the PKC exon (top panel). (*) Indicates bands quantified by a phosphorimager. Total SVA exonization was normalized to PKC-EGFP splicing within each respective lane and graphed (bottom panel).

SVA alternative splicing outcomes. An intronic truncated SVA is shown (middle). The SVA is truncated because these SVAs are still likely to be spliced. If SVAs are exonized, they will likely generate a truncated protein or subject the mRNA to nonsense-mediated decay due to the inclusion of SVA nonsense codons (top). If SVAs mimic an endogenous gene-trap, that is provide a 3′ SS followed by termination at the SVA or downstream polyA signal, this may result in truncated proteins, but more importantly the retrotransposition of exons.