title: Retrotransposition of marked SVA elements by human L1s in cultured cells.
abstract: Human retrotransposons generate structural variation and genomic diversity through ongoing retrotransposition and non-allelic homologous recombination. Cell culture retrotransposition assays have provided great insight into the genomic impact of retrotransposons, in particular, LINE-1(L1) and Alu elements; however, no such assay exists for the youngest active human retrotransposon, SINE-VNTR-Alu (SVA). Here we report the development of an SVA cell culture retrotransposition assay. We marked several SVAs with either neomycin or EGFP retrotransposition indicator cassettes. Engineered SVAs retrotranspose using L1 proteins supplemented in trans in multiple cell lines, including U2OS osteosarcoma cells where SVA retrotransposition is equal to that of an engineered L1. Engineered SVAs retrotranspose at 1-54 times the frequency of a marked pseudogene in HeLa HA cells. Furthermore, our data suggest a variable requirement for L1 ORF1p for SVA retrotransposition. Recovered engineered SVA insertions display all the hallmarks of LINE-1 retrotransposition and some contain 5' and 3' transductions, which are common for genomic SVAs. Of particular interest is the fact that four out of five insertions recovered from one SVA are full-length, with the 5' end of these insertions beginning within 5 nt of the CMV promoter transcriptional start site. This assay demonstrates that SVA elements are indeed mobilized in trans by L1. Previously intractable questions regarding SVA biology can now be addressed.
authors.affiliation: Cell and Molecular Biology Graduate Group, The University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
authors.forename: Dustin C
authors.initials: DC
authors.lastname: Hancks
authors.forename: John L
authors.initials: JL
authors.lastname: Goodier
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 / Q000235:genetics; D015152:Blotting, Northern; D002460:Cell Line; D045744:Cell Line, Tumor; D006367:HeLa Cells; D006801:Humans; D018598:Minisatellite Repeats / Q000235:genetics; D016133:Polymerase Chain Reaction; D018626:Retroelements / Q000235:genetics*; D020086:Short Interspersed Nucleotide Elements / Q000235:genetics; D024363:Transcription Initiation Site
chemical_list: D018626:Retroelements