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. 2011 Sep 1;20(17):3386-400.
doi: 10.1093/hmg/ddr245. Epub 2011 Jun 2.

Retrotransposition of marked SVA elements by human L1s in cultured cells

Dustin C Hancks  1 , John L Goodier, Prabhat K Mandal, Ling E Cheung, Haig H Kazazian Jr
Affiliations

Retrotransposition of marked SVA elements by human L1s in cultured cells

Dustin C Hancks et al. Hum Mol Genet. .

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.

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Figures

Figure 1.
Figure 1.
A cell-culture SVA retrotransposition assay. (A) A full-length ‘canonical' SVA in the human genome, with the individual domains in order from 5′ to 3′. (i) CCCTCT hexamer; (ii) the Alu-like domain consisting of two antisense-spliced Alu fragments and a sequence of unknown origin; (iii) VNTR; and (iv) SINE-R (env sequence and right LTR from an extinct HERV-K), terminating in a polyA tail (AAAn), with the entire insertion flanked by a TSD (black horizontal arrows). (B) The SVA.10 mneoI construct. A ‘master' SVA locus, SVA.10, from the SVAF1 (MAST2) subfamily containing both 5′ (5′ TR, Alu) and 3′ (Alu, 3′ TR) transductions (TR) marked with the mneoI retrotransposition cassette cloned into the pCEP-Pur plasmid backbone. (C) The rationale of the trans-complementation assay is illustrated. Only if the SVA containing the mneoI reporter undergoes a round of transcription, followed by reverse transcription and integration presumably mediated by a full-length L1 (shown), will the reading frame of the neomycin phosphotransferase reporter be restored, conferring G418 resistance (G418R). (D) G418R foci formation is observed in HeLa HA cells when SVA.10 mneoI is co-transfected with the highly active L1 driver construct, pcDNA.L1-RP. The mean number of clones per well ± SEM and the range of clones across the wells are displayed below. The number of wells (n) assayed for this experiment is shown. The retrotransposition frequency (mean number of clones/number of transfected cells) for SVA.10 mneoI is listed below the range.
Figure 2.
Figure 2.
Engineered SVA retrotransposition is mediated by human L1 proteins in HeLa HA cells. Different marked SVAs, ORF1 mneoI and Alu neoTet were co-transfected with various drivers (A–G) to determine the role of L1 proteins in SVA retrotransposition. All transfections were carried out in six-well plates with 1.5 µg of the corresponding ‘driver' plasmid and 0.5 µg of the corresponding ‘passenger' plasmid. Data are presented as the mean number of G418R foci per well ± SEM, with the number of replicates (n) below each mean. Where no data are presented, it means that the experiment was not carried out. ‘Hot' L1s, L1-RP and L1.3 mobilize engineered SVAs (A and B). Removal of the CMV and L1 promoter (5′ UTR) from pcDNA.L1-RP reduces SVA foci formation to background levels (C). Different drivers containing point mutations (D and G) or lacking ORF1 coding sequence (E and F) were co-transfected with SVA. (*) indicates the relative location of the engineered point mutation.
Figure 3.
Figure 3.
(A) Steady-state levels of spliced RNA from marked SVA constructs differ. HeLa cells were co-transfected with pcDNA.L1-RP and different SVA constructs. Northern analysis used a neo sense probe spanning the intron. A representative northern blot (10 µg of total RNA) is shown. Across the top are the names of the different SVA passenger constructs. Along the left side is a size standard in kilobases (kb). Below is the 28S rRNA loading control. The expected RNA lengths derived from the SVA constructs (kb) including spliced mneoI from the 5′ end of the element to the SV40 polyA signal in pCEP: SVA.10 mneoI = 5.5, SVA.10R mneoIΔ3tr = 4.9, SVA.2 mneoI = 3.5, Alu neoTet = 1.5. (B) Representative T-75 flasks of neo assays carried out are shown. Engineered SVAs or GFP mneoI were co-transfected with pcDNA.L1-RP or without driver plasmid (No Driver). Refer to Table 1 for foci counts and relative activity.
Figure 4.
Figure 4.
Engineered SVAs retrotranspose in multiple cell lines. (A) SVA.2 EGFP-positive foci at day 3 in U2OS cells are shown. SVAs are marked (x-axis) with the EGFP retrotransposition indicator cassette (76) and co-transfected with L1 drivers in HeLa HA (B), HEK 293T(C), U2OS (D) and 143B cells. All transfections were carried out in six-well plates (see Methods and Materials). Five days after transfection, cells were subjected to flow cytometry. Retrotransposition frequency was calculated as the number of events (EGFP-positive cells) relative to the number of cells transfected (y-axis) with the designated passenger plasmids (B–E). Events were gated on cells co-transfected with SVA.10 EGFP and pcDNA.L1-RP (D702Y). 99 RPS, 99 RPS EGFP Pur; JM111, JM111 RPS EGFP Pur; FL-L1, pcDNA.L1-RP (FL = L1/2 refers to a reduction in the amount of pcDNA.L1-RP transfected); ORF1, pcDNA.ORF1; ORF2, pcDNA.ORF2; ORF1/ORF2, co-transfection with pcDNA.ORF1 and pcDNA.ORF2 on separate plasmids. All transfections were performed in triplicate, except SVA.10R EGFP Δ3TR and SVA.10 EGFP/ORF1/ORF2 in HeLa HA cells. Where included, the mean %EGFP-positive cells is given. Error bars represent 1 standard deviation.
Figure 5.
Figure 5.
Engineered SVA insertions recovered from genomic DNA resemble SVA genomic insertions and display hallmarks of L1-mediated retrotransposition. Intron-spanning PCR was carried out on genomic DNA isolated from clonal cell lines derived from individual foci produced by different SVAs (A). Unspliced and spliced mneoI bands are indicated by black horizontal arrows. The 5′ and 3′′ ends for SVA.10 mneoI insertions recovered from individual foci (B–F). The genomic coordinates relative to the reference genome assembly (hg19/NCBI37) for each insertion are shown (right corner). The insertion-site nucleotide sequence consisting of the target-site duplication (bold letters), the L1 endonuclease cleavage site of the bottom strand (black vertical arrow) relative to the L1 endonuclease consensus cleavage site (5′-TTTT/a-3′) and 10 nucleotides 5′ and 3′ of the TSD are displayed. Each SVA insertion, including TSDs (black arrows), polyA signal and length of polyA tail, is diagrammed with individual domains annotated as described in Fig. 1B.
Figure 6.
Figure 6.
Engineered SVA insertions resemble SVA genomic insertions. (A) A consensus sequence, generated using WebLogo (86), for engineered SVAs insertion sites resembles the L1 endonuclease consensus cleavage site (5′-TTTT/a-3′) (–81) (Table 2). (B) An alignment of four SVA.10 mneoI insertions, containing 5′ transductions, relative to the SVA.10 mneoI plasmid sequence. The 3′ end of the CMV promoter in CEP labeled along with the known CMV transcriptional start site (black bent arrow). Note that the first base of each insertion is within 5 nt of the CMV transcriptional start site. For insertion 3, a non-templated G at the 5′ breakpoint is displayed as (G).
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