title: 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.
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Dustin C
authors.initials: DC
authors.lastname: Hancks
authors.forename: Prabhat K
authors.initials: PK
authors.lastname: Mandal
authors.forename: Ling E
authors.initials: LE
authors.lastname: Cheung
authors.forename: Haig H
authors.initials: HH
authors.lastname: Kazazian
mesh_terms: D020087:Alu Elements; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D018598:Minisatellite Repeats; D016254:Mutagenesis, Insertional; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D048348:Reverse Transcription
chemical_list: D022661:RNA, Untranslated; D049452:Green Fluorescent Proteins; D012194:RNA-Directed DNA Polymerase