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The minimal active human SVA retrotransposon requires only the 5'-hexamer and Alu-like domains.

ABSTRACT: RNA-based duplication mediated by reverse transcriptase (RT), a process termed retrotransposition, is ongoing in humans and is a source of significant inter- and perhaps intraindividual genomic variation. The long interspersed element 1 (LINE-1 or L1) ORF2 protein is the genomic source for RT activity required for mobilization of its own RNA in cis and other RNAs, such as SINE/variable-number tandem-repeat (VNTR)/Alu (SVA) elements, in trans. SVA elements are ~2-kb hominid-specific noncoding RNAs that have resulted in single-gene disease in humans through insertional mutagenesis or aberrant mRNA splicing. Here, using an SVA retrotransposition cell culture assay in U2OS cells, we investigated SVA domains important in L1-mediated SVA retrotransposition. Partial- and whole-domain deletions revealed that removal of either the Alu-like or SINE-R domain in the context of a full-length SVA has little to no effect, whereas removal of the CT hexamer or the VNTR domain can result in a 75% decrease in activity. Additional experiments demonstrate that the Alu-like fragment alone can retrotranspose at low levels while the addition of the CT hexamer can enhance activity as much as 2-fold compared to that of the full-length SVA. These results suggest that no SVA domain is essential for retrotransposition in U2OS cells and that the 5' end of SVA (hexamer and Alu-like domain) is sufficient for retrotransposition.

SUBMITTER: Hancks DC

PROVIDER: S-EPMC3486173 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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The minimal active human SVA retrotransposon requires only the 5'-hexamer and Alu-like domains.

Hancks Dustin C DC Mandal Prabhat K PK Cheung Ling E LE Kazazian Haig H HH

Molecular and cellular biology 20120924 22

RNA-based duplication mediated by reverse transcriptase (RT), a process termed retrotransposition, is ongoing in humans and is a source of significant inter- and perhaps intraindividual genomic variation. The long interspersed element 1 (LINE-1 or L1) ORF2 protein is the genomic source for RT activity required for mobilization of its own RNA in cis and other RNAs, such as SINE/variable-number tandem-repeat (VNTR)/Alu (SVA) elements, in trans. SVA elements are ~2-kb hominid-specific noncoding RNA ...[more]

PMID: 23007156

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Project description:Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of ?-dystroglycan (?-DG) and laminin binding by ?-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

Dataset Information

The minimal active human SVA retrotransposon requires only the 5'-hexamer and Alu-like domains.

Publications

The minimal active human SVA retrotransposon requires only the 5'-hexamer and Alu-like domains.

Similar Datasets

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