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1.
Nucleic Acids Res ; 52(13): 7761-7779, 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-38850156

RESUMEN

Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1) ORF2-encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 (Alu-permissive) strains, but not in HeLa-JVM or HeLa-H1 (Alu-nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA-derived SINEs and tRNA-derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR-Alu (SVA) element, and an L1 ORF1-containing mRNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu-permissive and Alu-nonpermissive HeLa strains, suggesting that 7SL- and tRNA-derived SINEs use strategies to 'hijack' L1 ORF2p that are distinct from those used by SVA elements and ORF1-containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu-permissive and Alu-nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu-nonpermissive HeLa strains.


Asunto(s)
Elementos Alu , Elementos de Nucleótido Esparcido Largo , Humanos , Células HeLa , Elementos Alu/genética , Elementos de Nucleótido Esparcido Largo/genética , Elementos de Nucleótido Esparcido Corto/genética , Animales , Retroelementos/genética , ARN/genética , ARN/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Pez Cebra/genética
2.
Proc Natl Acad Sci U S A ; 116(41): 20612-20622, 2019 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-31548405

RESUMEN

Long interspersed element-1 (LINE-1 or L1) amplifies via retrotransposition. Active L1s encode 2 proteins (ORF1p and ORF2p) that bind their encoding transcript to promote retrotransposition in cis The L1-encoded proteins also promote the retrotransposition of small-interspersed element RNAs, noncoding RNAs, and messenger RNAs in trans Some L1-mediated retrotransposition events consist of a copy of U6 RNA conjoined to a variably 5'-truncated L1, but how U6/L1 chimeras are formed requires elucidation. Here, we report the following: The RNA ligase RtcB can join U6 RNAs ending in a 2',3'-cyclic phosphate to L1 RNAs containing a 5'-OH in vitro; depletion of endogenous RtcB in HeLa cell extracts reduces U6/L1 RNA ligation efficiency; retrotransposition of U6/L1 RNAs leads to U6/L1 pseudogene formation; and a unique cohort of U6/L1 chimeric RNAs are present in multiple human cell lines. Thus, these data suggest that U6 small nuclear RNA (snRNA) and RtcB participate in the formation of chimeric RNAs and that retrotransposition of chimeric RNA contributes to interindividual genetic variation.


Asunto(s)
Células Madre Embrionarias/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias/genética , Células-Madre Neurales/metabolismo , ARN Nuclear Pequeño/genética , ARN/genética , Retroelementos/genética , Células HeLa , Humanos , Seudogenes , ARN/química , ARN Mensajero/química , ARN Mensajero/genética , ARN Nuclear Pequeño/química
3.
PLoS Biol ; 16(3): e2003067, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29505568

RESUMEN

Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5' untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5'UTR or 5'UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5'UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5'UTR and 5'UTR/ORF1 SpIREs lack Cis-acting transcription factor binding sites and exhibit reduced promoter activity. The 5'UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5'UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary "dead-ends" in the L1 retrotransposition process, mutations within the L1 5'UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation.


Asunto(s)
Evolución Molecular , Genoma Humano , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Células HeLa , Humanos , Polimorfismo Genético , Regiones Promotoras Genéticas , Empalme del ARN , ARN Mensajero/metabolismo
4.
PLoS Genet ; 11(5): e1005121, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25951186

RESUMEN

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements.


Asunto(s)
Elementos Alu , Elementos de Nucleótido Esparcido Largo/genética , Proteínas de Unión al ARN/metabolismo , Retroelementos , Animales , Cromatografía Liquida , Células HeLa , Humanos , Inmunoprecipitación , Ratones , Sistemas de Lectura Abierta , Plásmidos/genética , Plásmidos/metabolismo , Proteínas de Unión al ARN/genética , Espectrometría de Masas en Tándem , Pez Cebra
5.
Nucleic Acids Res ; 42(6): 3803-20, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24371271

RESUMEN

Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NΔ385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci. Finally, siRNA-mediated depletion of endogenous RNase L in a human ovarian cancer cell line (Hey1b) increased the levels of L1 retrotransposition by ∼2-fold. Together, these data suggest that RNase L might function as a suppressor of structurally distinct retrotransposons.


Asunto(s)
Endorribonucleasas/metabolismo , Genes de Partícula A Intracisternal , Elementos de Nucleótido Esparcido Largo , Animales , Línea Celular Tumoral , Células HeLa , Humanos , Ratones , ARN Mensajero/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo
6.
Proc Natl Acad Sci U S A ; 108(51): 20345-50, 2011 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-21940498

RESUMEN

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.


Asunto(s)
Elementos de Nucleótido Esparcido Largo/genética , ADN Polimerasa Dirigida por ARN/genética , Telomerasa/genética , Animales , Células CHO , Dominio Catalítico , Cricetinae , Cricetulus , Células HeLa , Humanos , Modelos Genéticos , Mutación , Sistemas de Lectura Abierta , Retroelementos/genética , Telómero/ultraestructura
7.
bioRxiv ; 2024 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-38746229

RESUMEN

Alu elements are non-autonomous Short INterspersed Elements (SINEs) derived from the 7SL RNA gene that are present at over one million copies in human genomic DNA. Alu mobilizes by a mechanism known as retrotransposition, which requires the Long INterspersed Element-1 (LINE-1 or L1) ORF2 -encoded protein (ORF2p). Here, we demonstrate that HeLa strains differ in their capacity to support Alu retrotransposition. Human Alu elements retrotranspose efficiently in HeLa-HA and HeLa-CCL2 ( Alu -permissive) strains, but not in HeLa-JVM or HeLa-H1 ( Alu -nonpermissive) strains. A similar pattern of retrotransposition was observed for other 7SL RNA -derived SINEs and tRNA -derived SINEs. In contrast, mammalian LINE-1s, a zebrafish LINE, a human SINE-VNTR - Alu ( SVA ) element, and an L1 ORF1 -containing messenger RNA can retrotranspose in all four HeLa strains. Using an in vitro reverse transcriptase-based assay, we show that Alu RNAs associate with ORF2p and are converted into cDNAs in both Alu -permissive and Alu -nonpermissive HeLa strains, suggesting that 7SL - and tRNA -derived SINE RNAs use strategies to 'hijack' L1 ORF2p that are distinct from those used by SVA elements and ORF1 -containing mRNAs. These data further suggest ORF2p associates with the Alu RNA poly(A) tract in both Alu -permissive and Alu -nonpermissive HeLa strains, but that Alu retrotransposition is blocked after this critical step in Alu -nonpermissive HeLa strains.

8.
PLoS Genet ; 6(10)2010 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-20949108

RESUMEN

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1-encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.


Asunto(s)
Elementos de Nucleótido Esparcido Largo/genética , Sistemas de Lectura Abierta/genética , Ribonucleoproteínas/genética , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Citoplasma/metabolismo , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Mutagénesis Insercional , Mutación , Plásmidos/genética , ARN/metabolismo , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/metabolismo , Transfección
9.
Sci Data ; 10(1): 813, 2023 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-37985666

RESUMEN

Somatic mosaicism is defined as an occurrence of two or more populations of cells having genomic sequences differing at given loci in an individual who is derived from a single zygote. It is a characteristic of multicellular organisms that plays a crucial role in normal development and disease. To study the nature and extent of somatic mosaicism in autism spectrum disorder, bipolar disorder, focal cortical dysplasia, schizophrenia, and Tourette syndrome, a multi-institutional consortium called the Brain Somatic Mosaicism Network (BSMN) was formed through the National Institute of Mental Health (NIMH). In addition to genomic data of affected and neurotypical brains, the BSMN also developed and validated a best practices somatic single nucleotide variant calling workflow through the analysis of reference brain tissue. These resources, which include >400 terabytes of data from 1087 subjects, are now available to the research community via the NIMH Data Archive (NDA) and are described here.


Asunto(s)
Trastornos Mentales , Humanos , Trastorno del Espectro Autista/genética , Encéfalo , Genómica , Mosaicismo , Genoma Humano , Trastornos Mentales/genética
10.
Genome Biol ; 22(1): 92, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33781308

RESUMEN

BACKGROUND: Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS: Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS: This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.


Asunto(s)
Encéfalo/metabolismo , Estudios de Asociación Genética , Variación Genética , Alelos , Mapeo Cromosómico , Biología Computacional/métodos , Estudios de Asociación Genética/métodos , Genómica/métodos , Células Germinativas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Especificidad de Órganos/genética , Polimorfismo de Nucleótido Simple
11.
Methods Mol Biol ; 1400: 139-56, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26895052

RESUMEN

The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Elementos Alu , Expresión Génica , Células HeLa , Humanos , Retroelementos , Transfección
12.
Microbiol Spectr ; 3(2): MDNA3-0061-2014, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26104698

RESUMEN

Transposable elements have had a profound impact on the structure and function of mammalian genomes. The retrotransposon Long INterspersed Element-1 (LINE-1 or L1), by virtue of its replicative mobilization mechanism, comprises ∼17% of the human genome. Although the vast majority of human LINE-1 sequences are inactive molecular fossils, an estimated 80-100 copies per individual retain the ability to mobilize by a process termed retrotransposition. Indeed, LINE-1 is the only active, autonomous retrotransposon in humans and its retrotransposition continues to generate both intra-individual and inter-individual genetic diversity. Here, we briefly review the types of transposable elements that reside in mammalian genomes. We will focus our discussion on LINE-1 retrotransposons and the non-autonomous Short INterspersed Elements (SINEs) that rely on the proteins encoded by LINE-1 for their mobilization. We review cases where LINE-1-mediated retrotransposition events have resulted in genetic disease and discuss how the characterization of these mutagenic insertions led to the identification of retrotransposition-competent LINE-1s in the human and mouse genomes. We then discuss how the integration of molecular genetic, biochemical, and modern genomic technologies have yielded insight into the mechanism of LINE-1 retrotransposition, the impact of LINE-1-mediated retrotransposition events on mammalian genomes, and the host cellular mechanisms that protect the genome from unabated LINE-1-mediated retrotransposition events. Throughout this review, we highlight unanswered questions in LINE-1 biology that provide exciting opportunities for future research. Clearly, much has been learned about LINE-1 and SINE biology since the publication of Mobile DNA II thirteen years ago. Future studies should continue to yield exciting discoveries about how these retrotransposons contribute to genetic diversity in mammalian genomes.


Asunto(s)
Variación Genética , Genoma , Elementos de Nucleótido Esparcido Largo , Recombinación Genética , Elementos de Nucleótido Esparcido Corto , Animales , Enfermedades Genéticas Congénitas , Humanos , Mamíferos
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