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. 2005 Sep;25(17):7780-95.
doi: 10.1128/MCB.25.17.7780-7795.2005.

Multiple fates of L1 retrotransposition intermediates in cultured human cells

Nicolas Gilbert  1 , Sheila Lutz, Tammy A Morrish, John V Moran
Affiliations

Multiple fates of L1 retrotransposition intermediates in cultured human cells

Nicolas Gilbert et al. Mol Cell Biol. 2005 Sep.

Abstract

LINE-1 (L1) retrotransposons comprise approximately 17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of approximately 1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome.

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Figures

FIG. 1.
FIG. 1.
Simple sequence alterations at the 5′ genomic DNA/L1 junction. A. Rationale of the assay. The 3′ UTR of a human RC-L1 was tagged with a reporter cassette designed to detect retrotransposition events. Open rectangles indicate L1 ORF1 and L1 ORF2, respectively. The relative positions of the endonuclease (EN), RT, and cysteine-rich domains (C) are indicated. The position of the L1 promoter (P) and the simian virus 40 late polyadenylation signal (pA) needed for L1 expression also are indicated. The mneoI gene and ColE1 bacterial origin of replication are indicated by light and dark gray rectangles, respectively. The relative positions of the prokaryotic/eukaryotic promoter (P′) and the thymidine kinase polyadenylation signal (A′) required for reporter gene expression also are shown. The mneoI gene is interrupted by an intron (γ-globin intron 2) in the same transcriptional orientation as the L1. SD and SA indicate the splice donor and splice acceptor sites, respectively. This arrangement ensures that a functional NEO transcript will be translated only following L1 retrotransposition. The putative structure of a resultant retrotransposition event that confers G418 resistance (G418r) to HeLa cells is shown at the bottom. Horizontal arrows flanking the resultant insertion indicate the TSD. Black lines flanking the insertion indicate HeLa genomic DNA, and RE represents the cleavage site for the restriction enzyme present in flanking genomic DNA. B. Extra sequences at the 5′ genomic DNA/L1 junction. The top center panel indicates a Y-branched L1 retrotransposition intermediate following the initial stages of TPRT. A variety of sequence alterations were found at the 5′ genomic DNA/L1 junction including the addition of single “untemplated” nucleotides (indicated by the “N” in the bottom left panel), the formation of a palindromic repeat (indicated by the arrows in the bottom middle panel), and the addition of “filler” DNA (indicated by the open rectangle in the bottom right panel).
FIG. 2.
FIG. 2.
cDNA additions at the 5′ genomic DNA/L1 junction. A. A processed pseudogene/L1 chimera. Insertion 19 was accompanied by the addition of a 5′-truncated cDNA copy of hnRNP H1 mRNA, which is located in the opposite transcriptional orientation of the L1. The undulating and straight gray lines represent hnRNP H1 mRNA and hnRNP H1 minus-strand cDNA, respectively. The undulating and straight black lines represent L1 mRNA and minus-strand L1 cDNA, respectively. Recombination between the resultant cDNAs resulted in a genomic deletion of approximately 14.1 kb. The structure of the hnRNP H1/L1 chimera is shown at the bottom of the figure. The striped and black rectangles indicate the integrated hnRNP H1 and L1 cDNAs, respectively. Double black lines indicate flanking genomic DNA. “An” indicates the poly(A) tail at the ends of the hnRNP H1 and L1 cDNAs. Small arrows indicate the positions of PCR primers used to characterize the insertion. B. A U6/L1 chimera. Insertion 20 was accompanied by the addition of a full-length U6 cDNA copy in the same transcriptional orientation as the L1. The undulating and straight black lines represent L1 mRNA and minus-strand L1 cDNA, respectively. The gray undulating line indicates U6 snRNA. The structure of the resultant chimera is shown at the bottom of the figure. The striped and black rectangles indicate the integrated U6 and L1 cDNAs, respectively. Double black lines indicate flanking genomic DNA. “An” indicates the poly(A) tail at the end of the L1 cDNA. Horizontal arrows indicate TSDs that flank the chimera.
FIG. 3.
FIG. 3.
Chimeric L1 insertions associated with genomic deletions. Insertions 60, 61, and 62 (A, B, and C, respectively) resulted in the formation of chimeric L1s and the concomitant deletion of target site nucleotides. Each insertion initiated by TPRT then likely was joined to target site DNA by single-strand annealing (the process is depicted in full in panel A). The undulating and straight black lines represent L1 mRNA and minus-strand L1 cDNA, respectively. The gray rectangles represent endogenous L1s, and “Am” represents the position of their poly(A) tails. The upward-pointing open-headed arrow indicates the L1 integration site. The gray/black-shaded rectangle indicates the resultant chimeric L1. “An” indicates the poly(A) tail at the end of the newly integrated L1. Small horizontal arrows indicate the positions of PCR primers used to characterize the insertion. The sizes of the target site deletions (in base pairs) are indicated in each panel.
FIG. 4.
FIG. 4.
Chimeric L1 insertions associated with genomic duplications. Insertions 66, 67, and 99 resulted in the formation of chimeric L1s and the duplication of an intrachromosomal segment of DNA. Each insertion initiated by TPRT and likely was repaired by synthesis-dependent strand annealing. The undulating and straight black lines represent L1 mRNA and minus-strand L1 cDNA, respectively. In event 66 (A), twin priming resulted in cDNA synthesis using the 3′-OH present at the top and bottom strands of the target site. The bottom-strand cDNA then used an endogenous L1 located ∼115 kb upstream of the insertion site (light blue rectangle) as a template for SDSA. As a result the newly integrated L1 was joined to the endogenous L1 as well as its flanking DNA (green line). Resolution of the intermediate resulted in the inversion/duplication of a 604- to 626-bp segment of DNA and the formation of a chimeric L1. The entire insertion is flanked by a 15-bp TSD (indicated as in Fig. 1). In event 67 (B), twin priming resulted in two L1 cDNAs using the 3′-OH present at the top and bottom strands of the target site. The top-strand cDNA then used an endogenous L1 located ∼119 kb upstream of the insertion site (light blue rectangle) as a template for SDSA. As a result the newly integrated L1 cDNA was covalently joined to the endogenous L1 as well as its flanking DNA (green line). Resolution of the intermediate resulted in the duplication of a 674- to 720-bp segment of DNA and the formation of a chimeric L1. The entire insertion is flanked by a 14-bp TSD (indicated as in Fig. 1). In event 99 (C), TPRT resulted in the initiation of L1 cDNA synthesis. The resultant cDNA then used an endogenous L1 located ∼300 kb downstream of the insertion site (light blue rectangle) as a template for SDSA. As a result the newly integrated L1 cDNA was covalently joined to the endogenous L1 (green line). Resolution of the intermediate resulted in the duplication of a 475- to 559-bp segment of L1 DNA and the formation of a chimeric L1. The entire insertion is flanked by a 159-bp TSD (indicated as in Fig. 1). The large horizontal black arrows indicate the transcriptional orientation of new inserted L1 fragments. The vertical bar is a schematic of the junction of the inverted fragments. In the three cases, the presence of discriminating single nucleotide polymorphisms between the engineered L1 and the endogenous L1 was used to determine the size ranges of the duplicated L1 fragments.
FIG. 4.
FIG. 4.
Chimeric L1 insertions associated with genomic duplications. Insertions 66, 67, and 99 resulted in the formation of chimeric L1s and the duplication of an intrachromosomal segment of DNA. Each insertion initiated by TPRT and likely was repaired by synthesis-dependent strand annealing. The undulating and straight black lines represent L1 mRNA and minus-strand L1 cDNA, respectively. In event 66 (A), twin priming resulted in cDNA synthesis using the 3′-OH present at the top and bottom strands of the target site. The bottom-strand cDNA then used an endogenous L1 located ∼115 kb upstream of the insertion site (light blue rectangle) as a template for SDSA. As a result the newly integrated L1 was joined to the endogenous L1 as well as its flanking DNA (green line). Resolution of the intermediate resulted in the inversion/duplication of a 604- to 626-bp segment of DNA and the formation of a chimeric L1. The entire insertion is flanked by a 15-bp TSD (indicated as in Fig. 1). In event 67 (B), twin priming resulted in two L1 cDNAs using the 3′-OH present at the top and bottom strands of the target site. The top-strand cDNA then used an endogenous L1 located ∼119 kb upstream of the insertion site (light blue rectangle) as a template for SDSA. As a result the newly integrated L1 cDNA was covalently joined to the endogenous L1 as well as its flanking DNA (green line). Resolution of the intermediate resulted in the duplication of a 674- to 720-bp segment of DNA and the formation of a chimeric L1. The entire insertion is flanked by a 14-bp TSD (indicated as in Fig. 1). In event 99 (C), TPRT resulted in the initiation of L1 cDNA synthesis. The resultant cDNA then used an endogenous L1 located ∼300 kb downstream of the insertion site (light blue rectangle) as a template for SDSA. As a result the newly integrated L1 cDNA was covalently joined to the endogenous L1 (green line). Resolution of the intermediate resulted in the duplication of a 475- to 559-bp segment of L1 DNA and the formation of a chimeric L1. The entire insertion is flanked by a 159-bp TSD (indicated as in Fig. 1). The large horizontal black arrows indicate the transcriptional orientation of new inserted L1 fragments. The vertical bar is a schematic of the junction of the inverted fragments. In the three cases, the presence of discriminating single nucleotide polymorphisms between the engineered L1 and the endogenous L1 was used to determine the size ranges of the duplicated L1 fragments.
FIG. 5.
FIG. 5.
A possible interchromosomal translocation generated upon L1 retrotransposition. Green and black lines indicate the structures of the preintegration sites on chromosomes 4 and 6, respectively. The insertion initiated at an integration site that map to chromosome 6 by endonuclease-dependent TPRT. The undulating and straight black lines represent L1 mRNA and minus-strand L1 cDNA, respectively. The resultant insertion is shown at the bottom of the figure. The filled black rectangle indicates the newly integrated L1. The red line indicates a 336- to 340-bp segment of DNA derived from chromosome 3. Small arrows indicate the positions of PCR primers used to characterize the insertion. PCR demonstrated that the respective junction sequences were not present in naïve HeLa cells and that the insertion is flanked by at least 6 kb of genomic DNA that maps to chromosomes 4 and 6, respectively.
FIG. 6.
FIG. 6.
Insertions at atypical integration sites. Insertions 22, 64, and 65 (A, B, and C, respectively) occurred at sequences that do not resemble an L1 EN consensus cleavage site. Each event resulted in the deletion of target site nucleotides, and the sizes of the deletions are indicated below the respective preintegration sites. A 55- to 59-bp MaLR sequence is located at the 5′ genomic DNA/L1 junction in insertion 22 (depicted by a white box). An endogenous L1 (gray rectangle) was completely replaced by the newly integrated L1 in insertion 64, and three untemplated base pairs are present at the L1/3′ genomic DNA junction. A 292- to 527-bp segment of endogenous L1 (gray rectangle) is located at the 5′ genomic DNA/L1 junction in insertion 65. The origin of this DNA segment remains unknown. Small arrows indicate the positions of PCR primers used to characterize the insertions.
FIG. 7.
FIG. 7.
Misincorporation error rate of the L1 RT. The binomial distribution was used to calculate the percentage of full-length L1s that are faithful copies of their respective progenitor element. The calculations are based on a base misincorporation rate of 1 error/6,584 bp (15 errors in 98,758 bases of L1 sequenced).
FIG. 8.
FIG. 8.
Competing pathways for L1 retrotransposition in cultured human cells. L1 retrotransposition can be initiated in two ways. The major pathway uses L1 EN to initiate TPRT (left panel). A secondary pathway could use a DNA lesion to initiate reverse transcription (right panel) (56). We propose that the resultant cDNA intermediate can be resolved using distinct DNA repair pathways (see Discussion). “Canonical” retrotransposition (left panel) results in the formation of L1 insertions with standard hallmarks of TPRT. “Abortive” retrotransposition (right panel) can lead to a variety of structures, depending on the DNA repair pathway that is utilized to resolve the L1 cDNA intermediates. The balance between “conventional” and “abortive” retrotransposition likely depends on the cellular milieu. Conventional retrotransposition may be more apparent in germ cells (depicted by a large horizontal gray arrow over a thin light gray arrow), whereas “abortive” retrotransposition may be more apparent in transformed cultured cells (depicted with equally sized gray horizontal arrows).
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