Full-Length Human L1 Insertions Retain the Capacity for High Frequency Retrotransposition in Cultured Cells

Kimberland, Michelle L.; Divoký, Vladimír; Prchal, Josef T.; Schwahn, Uwe; Berger, Wolfgang; Kazazian, Haig H.

doi:10.1093/hmg/8.8.1557

Cited by 153 publications

(176 citation statements)

References 20 publications

Supporting

Mentioning

170

Contrasting

Order By: Relevance

“…By comparison, cells transfected with the control plasmid pEGFP-N1, which does not contain any retrotransposon sequences, exhibited a steady decrease in EGFP expression over the course of the assay (from 97.2%, 58.8%, and 45.9%, respectively, in 293T, Gli36, and Hep3B cells on day 1 of the assay, to Ͻ0.5% in all cell lines by 3 weeks after transfection; data not shown). These data indicated that the A͞RT hybrid sequence in the plasmid encoded a functional L1 that could undergo retrotransposition after cell transfection at efficiencies comparable with those reported previously for L1 RP (13,15).…”

Section: Resultssupporting

confidence: 87%

See 1 more Smart Citation

L1 retrotransposition in nondividing and primary human somatic cells

Kubo¹,

Seleme

Soifer

et al. 2006

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

169

156

View full text Add to dashboard Cite

Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and͞or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3 -azido-3 -deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G 1͞S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G 0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.adenovirus ͉ LINE-1 ͉ quiescent cell ͉ hybrid vector R etrotransposons are mobile elements that insert into new genomic locations by reverse transcription of an RNA intermediate. Human long interspersed element-1 (L1) elements (1, 2) are non-LTR retrotransposons that comprise Ͼ17% of the human genome. The vast majority (Ͼ99%) of L1s are inactive because of point mutations, truncations, and other rearrangements; however, it is estimated that the average human diploid genome contains Ϸ80-100 retrotransposition-competent (RC)-L1s (3). RC-L1s have played and continue to play a significant role in shaping the genome through insertional mutagenesis, nonallelic recombination, and by trans mobilization of non-L1 RNAs (2, 4, 5).L1 retrotransposition requires transcription of L1 RNA, its transport to the cytoplasm, and translation of its two ORFs (ORF1 and ORF2). Both L1-encoded proteins (ORF1p and ORF2p) are thought to preferentially associate with their own encoding RNA (''cis preference'') to form a ribonucleoprotein particle (RNP) (6, 7), which is a proposed retrotransposition intermediate (8,9). The L1 RNP must access the nucleus, where the L1 endonuclease cleaves genomic DNA at a degenerate consensus sequence (5Ј-TTTT͞A and variant sequences) to liberate a 3Ј hydroxyl residue that is subsequently used by the L1 reverse transcriptase (RT) as a primer to copy the L1 sequence in situ, a process termed ''targetprimed reverse transcription'' (2, 10). The resultant L1 cDNA then is joined to target DNA, leading to typical L1 structural hallmarks [5Ј truncations and͞or internal inversions, 3Ј poly (A) tail, and target-site duplications (TSDs)]. Although a putative nucleolar localization signal has been identified in ORF2p (11), it still remains unclear whether the L1 RNP crosses an intact nuclear membrane or whether its entry requires mitotic nuclear envelope breakdown.We previously developed a hybrid vector system consisting of a high-capacity, helper-dependent adenovirus vector encoding a human RC-L1 element (L1.3) tagged with a neomycin-resistance (neo R ) retrotransposition indicator ...

show abstract

Section: Resultssupporting

confidence: 87%

“…The retrotransposition capacity of L1 RP is roughly 3-fold that of L1.3 (13,15), the element used in our first-generation A͞RT system (12). Moreover, because the L1 promoter (residing in the L1 5Ј UTR) is only active in certain cell types (14,16,17), we augmented the expression of L1 RP with the mouse pgk promoter.…”

Section: Resultsmentioning

confidence: 99%

L1 retrotransposition in nondividing and primary human somatic cells

Kubo¹,

Seleme

Soifer

et al. 2006

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

169

156

View full text Add to dashboard Cite

show abstract

“…There are only five known full-length L1s involved in de novo human retrotransposition (17)(18)(19)(20)(21). Although all have been assayed for retrotransposition activity (2,19,(22)(23)(24)(25), we repaired L1.2A (20) and compared the activity of all five in the context of our 82 L1s. We found that four of these five disease-causing insertions had activities of 150%, 100%, 50%, and 39% of L1 RP , making them among the most active elements ever tested in cell culture (Fig.…”

Section: Disease-causing L1s Are As Active As the Hot L1smentioning

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 .…”

mentioning

confidence: 99%

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

Brouha

Schustak

Badge

et al. 2003

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

965

1,075

View full text Add to dashboard Cite

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.

show abstract

“…2b). In addition, we characterized the effect of miR-128 neutralization by quantifying colony formation by essentially the same methodology with a transcription-and-translation cassette based on the neomycin-resistance gene (G418) 35 instead of the luciferase gene (Fig. 2c).…”

Section: Identification Of Retrotransposition-repressor Mirsmentioning

confidence: 99%

miR-128 represses L1 retrotransposition by binding directly to L1 RNA

Hamdorf

Idica

Zisoulis

et al. 2015

Nat Struct Mol Biol

View full text Add to dashboard Cite

VOLUME 22 NUMBER 10 OCTOBER 2015 nature structural & molecular biology a r t i c l e s microRNAs (miRs) are a class of small (~22-nt) genomically encoded molecules that inhibit translational initiation and stimulate decay of mRNA targets 1,2 . miRs are transcribed by RNA polymerase II and processed by the RNase III enzymes-Dicer and Drosha with its binding partner, DGCR8-to produce short double-stranded RNAs in the nucleus. One strand associates with the Argonaute (Ago) protein, thus forming the miR-mediated silencing complex (miRISC). miRs guide the pairing of miRISC, with imperfect complementarity, to sequences in target mRNAs, thus resulting in their subsequent destabilization and translational repression of the target 3 . The 'seed sequence' , at nucleotides 2-8, is a key determinant for miRISC-target recognition 4,5 . Recent data have shown that 35-40% of miR-binding sites are found in 3′ untranslated regions (UTRs), 40-50% in coding regions and <5% in 5′-UTR regions of mRNAs 6,7 . More than 60% of the human transcriptome has been predicted to be under miR regulation, thus making this post-transcriptional control pathway as important as protein pathways in the regulation of cell functions 2 . It is clear that miRs have essential roles in regulating diverse functions in normal and diseased cells 8,9 .L1 belongs to the most abundant class of autonomous transposable elements 10 . Human L1 contains two open reading frames, ORF1 and ORF2, which encode a protein with RNA-binding and nucleotide acid-chaperone activity (ORF1) 11 and a protein with endonuclease and reverse-transcriptase activities (ORF2) 12-15 , respectively. L1 mobilizes replicatively from one location in the genome to another by a 'copy-and-paste' mechanism, and it has been proposed to be a remnant of an ancient retrovirus 12,16 . Active and inactive L1s have been implicated in the evolution of mammalian genomes and are linked to cell-based diseases, including cancer [17][18][19] . In addition, somatic L1 insertions are biased toward regions of cancer-specific DNA hypomethylation, thus suggesting that L1 insertions may provide a selective advantage during tumorigenesis 20 . Mechanisms that operate at different levels in gene-expression hierarchies have been selected to control transposition-mediated mutagenesis and mitigate the potential negative effects of newly inserted elements. In germ cells, a specific small-RNA subtype (piwi-interacting RNAs (piRNAs)) efficiently counteracts L1 activity, but these RNAs are not expressed in nongerm cells 21,22 . Somatic cells attenuate element mobilization by DNA methylation of the L1 promoter 23 . Other methods of regulation are mediated by APOBEC proteins 24,25 , microprocessor interactions 26 and Ago-mediated RNA interference in mouse embryonic stem cells 27 . L1-promoter silencing is greatly attenuated, and L1 transcription is reactivated in hypomethylated cells, such as cancer cells and tumor-initiating cells, and is also reactivated during reprogramming [28][29][30] . Because miRs act as regulators of g...

show abstract

Full-Length Human L1 Insertions Retain the Capacity for High Frequency Retrotransposition in Cultured Cells

Cited by 153 publications

References 20 publications

L1 retrotransposition in nondividing and primary human somatic cells

L1 retrotransposition in nondividing and primary human somatic cells

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

miR-128 represses L1 retrotransposition by binding directly to L1 RNA

Contact Info

Product

Resources

About