Evidence Consistent with Human L1 Retrotransposition in Maternal Meiosis I

Brouha, Brook; Meischl, Christof; Ostertag, Eric; Boer, Martin de; Zhang, Yue; Neijens, H. J.; Roos, Dirk; Kazazian, Haig H.

doi:10.1086/341722

Cited by 109 publications

(134 citation statements)

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“…To increase the retrotransposition frequency in transgenic mice, we modified our previous transgene to include a more active L1 (L1 LRE3 ) (Brouha et al 2002(Brouha et al , 2003 tagged with a short markerless cassette, permitting PCR-based genotyping of smaller insertions (>400 bp) (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

“…Mless was cloned into the pJCC5-L1 LRE3 Brouha et al 2002) to create pBS-L1 LRE3 -mless. The L1 LRE3 -mless was then cloned into pCEP-Hsp70-2, a vector similar to pCEP4 (Invitrogen), but containing the mouse heat-shock protein 70-2 promoter (pHSP70-2, cloned from the mouse genome) (Dix et al 1996) instead of the CMV promoter to create the transgene used in this study.…”

Section: Transgene Constructionmentioning

confidence: 99%

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L1 integration in a transgenic mouse model

et al. 2005

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To study integration of the human LINE-1 retrotransposon (L1) in vivo, we developed a transgenic mouse model of L1 retrotransposition that displays de novo somatic L1 insertions at a high frequency, occasionally several insertions per mouse. We mapped 3Ј integration sites of 51 insertions by Thermal Asymmetric Interlaced PCR (TAIL-PCR). Analysis of integration locations revealed a broad genomic distribution with a modest preference for intergenic regions. We characterized the complete structures of 33 de novo retrotransposition events. Our results highlight the large number of highly truncated L1s, as over 52% (27/51) of total integrants were <1/3 the length of a full-length element. New integrants carry all structural characteristics typical of genomic L1s, including a number with inversions, deletions, and 5Ј-end microhomologies to the target DNA sequence. Notably, at least 13% (7/51) of all insertions contain a short stretch of extra nucleotides at their 5Ј end, which we postulate result from template-jumping by the L1-encoded reverse transcriptase. We propose a unified model of L1 integration that explains all of the characteristic features of L1 retrotransposition, such as 5Ј truncations, inversions, extra nucleotide additions, and 5Ј boundary and inversion point microhomologies.[Supplemental material is available online at www.genome.org.]The long interspersed nucleotide element-1 (L1) retrotransposon enjoyed tremendous evolutionary success in colonizing eukaryotic genomes (Kazazian Jr. 2004); its roughly 500,000 copies comprise ∼17% of human DNA (Lander et al. 2001). The full-length 6-kb L1 encodes a 5Ј UTR containing an internal promoter, two proteins-ORF1, a nucleic acid-binding protein with chaperone activity (Hohjoh andSinger 1996, 1997;Martin and Bushman 2001), and ORF2, a protein with endonuclease (EN) and reverse transcriptase (RT) activities (Mathias et al. 1991;Feng et al. 1996), and a 3Ј UTR ending with a poly(A) tail (Fig. 1A). Both ORF1 and ORF2 proteins are required for retrotransposition . Once transcribed and translated, L1 RNA is copied into the genome by target-primed reverse transcription (TPRT) (Luan et al. 1993;Cost et al. 2002). During TPRT, one strand of host DNA is cleaved by the EN to expose a free 3Ј-hydroxyl, which is then used by the RT as a primer in reverse transcription, copying the L1 RNA template directly into the host genome. To complete integration, the second strand of host DNA must be cleaved, RNA removed, the newly synthesized strand copied, and breaks resolved. The mechanism by which these later steps are accomplished is only beginning to be understood, with recent biochemical data from a related non-LTR retrotransposon R2 of the silkmoth, Bombyx mori, suggesting that a second R2 protein is involved in cleavage, RNA displacement, and synthesis of the second strand (Bibillo and Eickbush 2002a; Christensen and Eickbush 2005).Following essentially random integration, endogenous L1s are thought to be lost over time due to strong negative selection leading to their uneven ...

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Section: Resultsmentioning

confidence: 99%

Section: Transgene Constructionmentioning

confidence: 99%

L1 integration in a transgenic mouse model

et al. 2005

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“…In addition to its normal and evolutionary relevant replication niche in germline and early embryonic cells (26,27,(69)(70)(71), L1 also can be active in some somatic cells, including certain tumors and neuronal progenitor cells (19,20,23), as well as a consequence of aging (72). ORF1p competition for kinases in any of these cells could perturb signaling cascades.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of ORF1p is required for L1 retrotransposition

Cook

Jones

Furano

2015

Proc. Natl. Acad. Sci. U.S.A.

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Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.L 1 (or LINE-1) activity over the last ∼100 million years of primate evolution has generated ∼40% of the human genome (1, 2); thus, succeeding families of L1 elements are the main drivers of genetic expansion. These autonomously replicating elements convert their RNA transcripts and those of other genetic elements, particularly SINEs, into genomic DNA (3). A generic L1 element is 6-7 kb and contains the following: a 5′ UTR; ORF1, which encodes the coiled-coil mediated trimeric nucleic acid chaperone protein ORF1p; ORF2, which encodes a DNA endonuclease and reverse-transcriptase ORF2p; and a 3′ UTR terminated in a polyA sequence (reviewed in refs. 3 and 4). ORF1p, ORF2p, and L1 RNA form ribonucleoprotein particles (RNPs) that are likely intermediates in L1 retrotransposition (5-8). The L1-encoded proteins ORF1p and ORF2p are essential in cell culture-based retrotransposition assays (9) and in vitro assays using RNPs from cells transfected with L1 retrotransposition vectors (7). Although the role of ORF1p in retrotransposition is not known, mutations that affect its nucleic acid binding and chaperone activities can inactivate L1 (7, 9, 10).L1 activity can damage DNA (11), can generate genetic diversity and rearrangements (12-16), and is activated in certain tumors (17-19) and other somatic cells (20), including neuronal cells (21-23). Despite being deleterious (2...

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“…L1-containing bands from at least two separate PCRs were pooled, band-purified by using a GeneClean Spin Kit (Bio 101), and digested with NotI and BstZ17I (New England Biolabs). Fragments were swapped into pL1 RP -enhanced GFP (EGFP) (BstZ17I) (19) [full-length L1 RP tagged with the EGFP retrotransposition cassette; pL1 RP -EGFP (23), except modified to contain a BstZ17I site] by using T4 Ligase (New England Biolabs). At least four clones of each L1 except al356438 (two clones) were assayed for activity.…”

Section: Methodsmentioning

confidence: 99%

“…Two (L1 RP and L1 ␤-Thal ) are full-length disease-producing insertions (17,18), and three (L1.2, LRE2, and LRE3) are the likely progenitors of disease-producing insertions (19)(20)(21). When assayed for retrotransposition, L1 RP , L1 ␤-Thal , and LRE3, all of which were isolated from affected individuals or family members, are hot L1s (19,(22)(23)(24). Hot L1s are defined as showing at least one-third of the activity of L1 RP .…”

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confidence: 99%

Hot L1s account for the bulk of retrotransposition in the human population

Brouha

Schustak

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et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Although LINE-1 (long interspersed nucleotide element-1, L1) retrotransposons comprise 17% of the human genome, an exhaustive search of the December 2001 ''freeze'' of the haploid human genome working draft sequence (95% complete) yielded only 90 L1s with intact ORFs. We demonstrate that 38 of 86 (44%) L1s are polymorphic as to their presence in human populations. We cloned 82 (91%) of the 90 L1s and found that 40 of the 82 (49%) are active in a cultured cell retrotransposition assay. From these data, we predict that there are 80 -100 retrotransposition-competent L1s in an average human being. Remarkably, 84% of assayed retrotransposition capability was present in six highly active L1s (hot L1s). By comparison, four of five full-length L1s involved in recent human insertions had retrotransposition activity comparable to the six hot L1s in the human genome working draft sequence. Thus, our data indicate that most L1 retrotransposition in the human population stems from hot L1s, with the remaining elements playing a lesser role in genome plasticity.

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Evidence Consistent with Human L1 Retrotransposition in Maternal Meiosis I

Cited by 109 publications

References 35 publications

L1 integration in a transgenic mouse model

L1 integration in a transgenic mouse model

Phosphorylation of ORF1p is required for L1 retrotransposition

Hot L1s account for the bulk of retrotransposition in the human population

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