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 ϳ2-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.A pproximately one-third of the human genome (52) is derived from the direct (cis) or indirect (trans) reverse transcriptase (RT) activity of the long interspersed element 1 (LINE-1 or L1). A full-length active L1 (6.0 kb) (70) is the only autonomous retrotransposon in humans. It contains two nonoverlapping open reading frames (ORFs) (70), an ϳ900-bp 5= untranslated region (UTR), with promoter activity (75), and a short 3=-UTR. ORF1 encodes a 40-kDa RNA binding protein (ORF1p) (40) with chaperone activity in vitro (56), whereas ORF2 encodes a 150-kDa protein with demonstrated endonuclease (EN) (25) and RT (57) activities. Both ORF1p and ORF2p are required for retrotransposition (20, 61) of their encoding RNA, a phenomenon termed cis preference (20,23,61,82).Most human L1s are inactive due to 5= truncations, point mutations, or internal rearrangements; however, a subset, ϳ80 to 100 in any given individual, remain active (4, 8). These active L1 loci are the source for new L1 insertions and can provide the L1 proteins required to mobilize other RNAs (Alu [7,19], SVA elements [37,65,67], U6 [9,27,31], and processed pseudogenes [23,82]) in trans. Retrotransposition is ongoing in the human population (4,6,24,(41)(42)(43)46), with almost 100 cases of single-gene disease (5,11,39,83) caused by L1 (47), Alu (80), SVA (50, 65), or poly(A) insertions. L1-mediated insertions may be deleterious by disrupting mRNA expression (32, 76) of a specific gene (ϳ25 examples [39,78]) or mitigating nonallelic homologous recombination (18,35,71). New insertions may also contribute to somatic mosaicism (28,45,62,77), in particular, neuronal diversi...