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1.
Nucleic Acids Res ; 52(9): 5166-5178, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38647072

RESUMO

L1 elements are retrotransposons currently active in mammals. Although L1s are typically silenced in most normal tissues, elevated L1 expression is associated with a variety of conditions, including cancer, aging, infertility and neurological disease. These associations have raised interest in the mapping of human endogenous de novo L1 insertions, and a variety of methods have been developed for this purpose. Adapting these methods to mouse genomes would allow us to monitor endogenous in vivo L1 activity in controlled, experimental conditions using mouse disease models. Here, we use a modified version of transposon insertion profiling, called nanoTIPseq, to selectively enrich young mouse L1s. By linking this amplification step with nanopore sequencing, we identified >95% annotated L1s from C57BL/6 genomic DNA using only 200 000 sequencing reads. In the process, we discovered 82 unannotated L1 insertions from a single C57BL/6 genome. Most of these unannotated L1s were near repetitive sequence and were not found with short-read TIPseq. We used nanoTIPseq on individual mouse breast cancer cells and were able to identify the annotated and unannotated L1s, as well as new insertions specific to individual cells, providing proof of principle for using nanoTIPseq to interrogate retrotransposition activity at the single-cell level in vivo.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Genoma/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos Endogâmicos C57BL , Sequenciamento por Nanoporos/métodos , Retroelementos/genética , Análise de Sequência de DNA/métodos
2.
PLoS Genet ; 13(6): e1006837, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28586350

RESUMO

Long interspersed nuclear element-1s (LINE-1s, or L1s) are an active family of retrotransposable elements that continue to mutate mammalian genomes. Despite the large contribution of L1 to mammalian genome evolution, we do not know where active L1 particles (particles in the process of retrotransposition) are located in the cell, or how they move towards the nucleus, the site of L1 reverse transcription. Using a yeast model of LINE retrotransposition, we identified ESCRT (endosomal sorting complex required for transport) as a critical complex for LINE retrotransposition, and verified that this interaction is conserved for human L1. ESCRT interacts with L1 via a late domain motif, and this interaction facilitates L1 replication. Loss of the L1/ESCRT interaction does not impair RNP formation or enzymatic activity, but leads to loss of retrotransposition and reduced L1 endonuclease activity in the nucleus. This study highlights the importance of the ESCRT complex in the L1 life cycle and suggests an unusual mode for L1 RNP trafficking.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Elementos Nucleotídeos Longos e Dispersos , Membrana Celular/metabolismo , Células HeLa , Humanos , Ligação Proteica , Transporte Proteico , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
3.
Nucleic Acids Res ; 42(1): 396-416, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24101588

RESUMO

LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.


Assuntos
Citidina Desaminase/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Proteínas/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas de Transporte/análise , Citidina Desaminase/química , Citidina Desaminase/genética , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/enzimologia , DNA Helicases , Células HeLa , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Multimerização Proteica , Proteínas/análise , Proteínas/química , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA
4.
Nucleic Acids Res ; 40(21): 10866-77, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22977178

RESUMO

Non-long terminal repeat (non-LTR) retrotransposons are highly abundant elements that are present in chromosomes throughout the eukaryotic domain of life. The long interspersed nuclear element (LINE-1) (L1) clade of non-LTR retrotransposons has been particularly successful in mammals, accounting for 30-40% of human genome sequence. The current model of LINE retrotransposition, target-primed reverse transcription, culminates in a chromosomally integrated end product. Using a budding yeast model of non-LTR retrotransposition, we show that in addition to producing these 'classical', chromosomally integrated products, a fungal L1 clade member (Zorro3) can generate abundant, RNA-derived episomal products. Genetic evidence suggests that these products are likely to be formed via a variation of target-primed reverse transcription. These episomal products are a previously unseen alternative fate of LINE retrotransposition, and may represent an unexpected source for de novo retrotransposition.


Assuntos
DNA Circular/biossíntese , Elementos Nucleotídeos Longos e Dispersos , Candida albicans/genética , DNA Circular/química , DNA Fúngico/biossíntese , DNA Fúngico/química , Humanos , Mutação , Saccharomycetales/genética , Homologia de Sequência do Ácido Nucleico
5.
bioRxiv ; 2023 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-38014156

RESUMO

L1 elements are retrotransposons currently active in mammals. Although L1s are typically silenced in most normal tissues, elevated L1 expression is associated with a variety of conditions, including cancer, aging, infertility, and neurological disease. These associations have raised interest in the mapping of human endogenous de novo L1 insertions, and a variety of methods have been developed for this purpose. Adapting these methods to mouse genomes would allow us to monitor endogenous in vivo L1 activity in controlled, experimental conditions using mouse disease models. Here we use a modified version of transposon insertion profiling, called nanoTIPseq, to selectively enrich young mouse L1s. By linking this amplification step with nanopore sequencing, we identified >95% annotated L1s from C57BL/6 genomic DNA using only 200,000 sequencing reads. In the process, we discovered 82 unannotated L1 insertions from a single C57BL/6 genome. Most of these unannotated L1s were near repetitive sequence and were not found with short-read TIPseq. We used nanoTIPseq on individual mouse breast cancer cells and were able to identify the annotated and unannotated L1s, as well as new insertions specific to individual cells, providing proof of principle for using nanoTIPseq to interrogate retrotransposition activity at the single cell level in vivo .

6.
Genetics ; 181(1): 301-11, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18957700

RESUMO

Over one-third of human genome sequence is a product of non-LTR retrotransposition. The retrotransposon that currently drives this process in humans is the highly abundant LINE-1 (L1) element. Despite the ubiquitous nature of L1's in mammals, we still lack a complete mechanistic understanding of the L1 replication cycle and how it is regulated. To generate a genetically amenable model for non-LTR retrotransposition, we have reengineered the Zorro3 retrotransposon, an L1 homolog from Candida albicans, for use in the budding yeast Saccharomyces cerevisiae. We found that S. cerevisiae, which has no endogenous L1 homologs or remnants, can still support Zorro3 retrotransposition. Analysis of Zorro3 mutants and insertion structures suggest that this is authentic L1-like retrotransposition with remarkable resemblance to mammalian L1-mediated events. This suggests that S. cerevisiae has unexpectedly retained the basal host machinery required for L1 retrotransposition. This model will also serve as a powerful system to study the cell biology of L1 elements and for the genetic identification and characterization of cellular factors involved in L1 retrotransposition.


Assuntos
Elementos de DNA Transponíveis/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Endonucleases/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Moldes Genéticos
7.
Nature ; 429(6989): 314-8, 2004 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15152256

RESUMO

LINE-1 (L1) elements are retrotransposons that comprise large fractions of mammalian genomes. Transcription through L1 open reading frames is inefficient owing to an elongation defect, inhibiting the robust expression of L1 RNA and proteins, the substrate and enzyme(s) for retrotransposition. This elongation defect probably controls L1 transposition frequency in mammalian cells. Here we report bypassing this transcriptional defect by synthesizing the open reading frames of L1 from synthetic oligonucleotides, altering 24% of the nucleic acid sequence without changing the amino acid sequence. Such resynthesis led to greatly enhanced steady-state L1 RNA and protein levels. Remarkably, when the synthetic open reading frames were substituted for the wild-type open reading frames in an established retrotransposition assay, transposition levels increased more than 200-fold. This indicates that there are probably no large, rigidly conserved cis-acting nucleic acid sequences required for retrotransposition within L1 coding regions. These synthetic retrotransposons are also the most highly active L1 elements known so far and have potential as practical tools for manipulating mammalian genomes.


Assuntos
Engenharia Genética , Elementos Nucleotídeos Longos e Dispersos/genética , Recombinação Genética/genética , Células 3T3 , Animais , Sequência de Bases , Células HeLa , Humanos , Camundongos , Mutagênese/genética , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , Proteínas/genética , Transcrição Gênica/genética
8.
Nature ; 429(6989): 268-74, 2004 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15152245

RESUMO

LINE-1 (L1) elements are the most abundant autonomous retrotransposons in the human genome, accounting for about 17% of human DNA. The L1 retrotransposon encodes two proteins, open reading frame (ORF)1 and the ORF2 endonuclease/reverse transcriptase. L1 RNA and ORF2 protein are difficult to detect in mammalian cells, even in the context of overexpression systems. Here we show that inserting L1 sequences on a transcript significantly decreases RNA expression and therefore protein expression. This decreased RNA concentration does not result from major effects on the transcription initiation rate or RNA stability. Rather, the poor L1 expression is primarily due to inadequate transcriptional elongation. Because L1 is an abundant and broadly distributed mobile element, the inhibition of transcriptional elongation by L1 might profoundly affect expression of endogenous human genes. We propose a model in which L1 affects gene expression genome-wide by acting as a 'molecular rheostat' of target genes. Bioinformatic data are consistent with the hypothesis that L1 can serve as an evolutionary fine-tuner of the human transcriptome.


Assuntos
Regulação para Baixo/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Transcrição Gênica/genética , Animais , Sequência de Bases , Linhagem Celular , Biologia Computacional , Endonucleases/biossíntese , Endonucleases/genética , Evolução Molecular , Retroalimentação Fisiológica , Meia-Vida , Humanos , Mamíferos/genética , Camundongos , Modelos Genéticos , Fases de Leitura Aberta/genética , RNA Polimerase III/metabolismo , Estabilidade de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , DNA Polimerase Dirigida por RNA/biossíntese , DNA Polimerase Dirigida por RNA/genética
9.
Genesis ; 46(7): 373-83, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18615728

RESUMO

The synthetic L1 retrotransposon, ORFeus, is useful for probing mechanisms of L1 retrotransposition in vivo and for genome-wide mouse mutagenesis because of its high level of activity. To achieve controlled activation of ORFeus in mice, we constructed ORFeus(LSL), in which ORFeus coding sequences were separated from the promoter by a loxP-beta-geo-stop-loxP (LSL) cassette, and derived transgenic mouse lines containing single-copy ORFeus(LSL). We observed tissue-specific ORFeus activation by crossing ORFeus(LSL) to various Cre-expressing lines, specifically in the germ line or the pancreas, providing definite evidence that all host factors and machinery required posttranscriptionally for L1 retrotransposition are available in somatic tissues in living animals. Notably, the single-copy ORFeus transgene is about threefold more active per copy than a previously described multicopy ORFeus transgene in the germ line and even more active somatically. This conditional transgenic ORFeus mouse model should allow further exploration of posttranscriptional cellular requirements for L1 retrotransposition and facilitate the development of ORFeus mouse lines suitable for in vivo mutagenesis.


Assuntos
Regulação da Expressão Gênica/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos Transgênicos/genética , Transgenes/genética , Animais , Vetores Genéticos/genética , Integrases , Camundongos , Mutagênese
10.
Methods Mol Biol ; 1400: 131-7, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26895051

RESUMO

Long interspersed nuclear element (LINE) retrotransposons make up significant parts of mammalian genomes. They alter host genomes by direct mutagenesis through integration of new transposon copies, by mobilizing non-autonomous transposons, by changes in host gene activity due to newly integrated transposons and by recombination events between different transposon copies. As a consequence, LINEs can contribute to genetic disease. Simple model systems can be useful for the study of basic molecular and cellular biology of LINE retrotransposons. Here, we describe methods for the analysis of LINE retrotransposition in the well-established model organism Saccharomyces cerevisiae. The ability to follow retrotransposition in budding yeast opens up the possibility of performing systematic screens for evolutionarily conserved interactions between LINE retrotransposons and their host cells.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Saccharomyces cerevisiae/genética , Clonagem Molecular/métodos , Genoma Fúngico , Genômica/métodos , Reação em Cadeia da Polimerase/métodos , Transformação Genética
11.
Science ; 344(6179): 55-8, 2014 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-24674868

RESUMO

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.


Assuntos
Cromossomos Fúngicos , Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Sequência de Bases , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Fúngico/genética , Genes Fúngicos , Aptidão Genética , Genoma Fúngico , Instabilidade Genômica , Íntrons , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , RNA Fúngico/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Análise de Sequência de DNA , Deleção de Sequência , Transformação Genética
12.
Mob DNA ; 2(1): 2, 2011 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-21320307

RESUMO

Long interspersed elements, type 1(LINE-1, L1) are the most abundant and only active autonomous retrotransposons in the human genome. Native L1 elements are inefficiently expressed because of a transcription elongation defect thought to be caused by high adenosine content in L1 sequences. Previously, we constructed a highly active synthetic mouse L1 element (ORFeus-Mm), partially by reducing the nucleotide composition bias. As a result, the transcript abundance of ORFeus-Mm was greatly increased, and its retrotransposition frequency was > 200-fold higher than its native counterpart. In this paper, we report a synthetic human L1 element (ORFeus-Hs) synthesized using a similar strategy. The adenosine content of the L1 open reading frames (ORFs) was reduced from 40% to 27% by changing 25% of the bases in the ORFs, without altering the amino acid sequence. By studying a series of native/synthetic chimeric elements, we observed increased levels of full-length L1 RNA and ORF1 protein and retrotransposition frequency, mostly proportional to increased fraction of synthetic sequence. Overall, the fully synthetic ORFeus-Hs has > 40-fold more RNA but is at most only ~threefold more active than its native counterpart (L1RP); however, its absolute retrotransposition activity is similar to ORFeus-Mm. Owing to the elevated expression of the L1 RNA/protein and its high retrotransposition ability, ORFeus-Hs and its chimeric derivatives will be useful tools for mechanistic L1 studies and mammalian genome manipulation.

13.
Mob DNA ; 1(1): 15, 2010 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-20462415

RESUMO

Non-long terminal repeat (non-LTR) retrotransposons are present in most eukaryotic genomes. In some species, such as humans, these elements are the most abundant genome sequence and continue to replicate to this day, creating a source of endogenous mutations and potential genotoxic stress. This review will provide a general outline of the replicative cycle of non-LTR retrotransposons. Recent findings regarding the host regulation of non-LTR retrotransposons will be summarized. Finally, future directions of interest will be discussed.

14.
Proc Natl Acad Sci U S A ; 103(49): 18662-7, 2006 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-17124176

RESUMO

Long interspersed element type 1 (L1) retrotransposons are ubiquitous mammalian mobile elements and potential tools for in vivo mutagenesis; however, native L1 elements are relatively inactive in mice when introduced as transgenes. We have previously described a synthetic L1 element, ORFeus, containing two synonymously recoded ORFs relative to mouse L1. It is significantly more active for retrotransposition in cell culture than all native L1 elements tested. To study its activity in vivo, we developed a transgenic mouse model in which ORFeus expression was controlled by a constitutive heterologous promoter, and we established definitive evidence for ORFeus retrotransposition activity both in germ line and somatic tissues. Germ line retrotransposition frequencies resulting in 0.33 insertions per animal are seen among progeny of ORFeus donor element heterozygotes derived from a single founder, representing a >20-fold increase over native L1 elements. We observe somatic transposition events in 100% of the ORFeus donor-containing animals, and an average of 17 different insertions are easily recovered from each animal; modeling suggests that the number of somatic insertions per animal exceeds this number by perhaps several orders of magnitude. Nearly 200 insertions were precisely mapped, and their distribution in the mouse genome appears random relative to transcription units and guanine-cytosine content. The results suggest that ORFeus may be developed into useful tools for in vivo mutagenesis.


Assuntos
Elementos Nucleotídeos Longos e Dispersos/fisiologia , Recombinação Genética/fisiologia , Animais , Feminino , Células HeLa , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
15.
Bioessays ; 27(8): 775-84, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16015595

RESUMO

LINE-1 (L1) retrotransposons are replicating repetitive elements that, by mass, are the most-abundant sequences in the human genome. Over one-third of mammalian genomes are the result, directly or indirectly, of L1 retrotransposition. L1 encodes two proteins: ORF1, an RNA-binding protein, and ORF2, an endonuclease/reverse transcriptase. Both proteins are required for L1 mobilization. Apart from the obvious function of self-replication, it is not clear what other roles, if any, L1 plays within its host. The sheer magnitude of L1 sequences in our genome has fueled speculation that over evolutionary time L1 insertions may structurally modify endogenous genes and regulate gene expression. Here we provide a review of L1 replication and its potential functional consequences.


Assuntos
Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Retroelementos/genética , Alelos , Citoplasma/metabolismo , DNA/química , Epigênese Genética , Evolução Molecular , Éxons , Genoma , Humanos , Modelos Biológicos , Modelos Genéticos , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Recombinação Genética , Raios Ultravioleta
16.
Genome Res ; 15(8): 1073-8, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16024818

RESUMO

The L1 retrotransposon is the most highly successful autonomous retrotransposon in mammals. This prolific genome parasite may on occasion benefit its host through genome rearrangements or adjustments of host gene expression. In examining possible effects of L1 elements on host gene expression, we investigated whether a full-length L1 element inserted in the antisense orientation into an intron of a cellular gene may actually split the gene's transcript into two smaller transcripts: (1) a transcript containing the upstream exons and terminating in the major antisense polyadenylation site (MAPS) of the L1, and (2) a transcript derived from the L1 antisense promoter (ASP) that includes the downstream exons of the gene. Bioinformatic analysis and experimental follow-up provide evidence for this L1 "gene-breaking" hypothesis. We identified three human genes apparently "broken" by L1 elements, as well as 12 more candidate genes. Most of the inserted L1 elements in our 15 candidate genes predate the human/chimp divergence. If indeed split, the transcripts of these genes may in at least one case encode potentially interacting proteins, and in another case may encode novel proteins. Gene-breaking represents a new mechanism through which L1 elements remodel mammalian genomes.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica , Retroelementos/genética , Animais , Elementos Antissenso (Genética) , Sequência de Bases , Éxons , Etiquetas de Sequências Expressas , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Pan troglodytes/genética , Poliadenilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met , Receptores de Fatores de Crescimento/genética , Transcrição Gênica
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