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1.
Nat Commun ; 15(1): 3883, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38719805

RESUMEN

The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. L1 harms the cell by inserting new copies, generating DNA damage, and triggering inflammation. Therefore, L1 inhibition could be used to treat many diseases associated with these processes. Previous research has focused on inhibition of the L1 reverse transcriptase due to the prevalence of well-characterized inhibitors of related viral enzymes. Here we present the L1 endonuclease as another target for reducing L1 activity. We characterize structurally diverse small molecule endonuclease inhibitors using computational, biochemical, and biophysical methods. We also show that these inhibitors reduce L1 retrotransposition, L1-induced DNA damage, and inflammation reinforced by L1 in senescent cells. These inhibitors could be used for further pharmacological development and as tools to better understand the life cycle of this element and its impact on disease processes.


Asunto(s)
Endonucleasas , Elementos de Nucleótido Esparcido Largo , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Endonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Daño del ADN , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Senescencia Celular/efectos de los fármacos , Desoxirribonucleasa I
2.
EBioMedicine ; 103: 105133, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38677181

RESUMEN

BACKGROUND: Endogenous retroelements (EREs), including human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), comprise almost half of the human genome. Our previous studies of the interferome in the gut suggest potential mechanisms regarding how IFNb may drive HIV-1 gut pathogenesis. As ERE activity is suggested to partake in type 1 immune responses and is incredibly sensitive to viral infections, we sought to elucidate underlying interactions between ERE expression and gut dynamics in people living with HIV-1 (PLWH). METHODS: ERE expression profiles from bulk RNA sequencing of colon biopsies and PBMC were compared between a cohort of PLWH not on antiretroviral therapy (ART) and uninfected controls. FINDINGS: 59 EREs were differentially expressed in the colon of PLWH when compared to uninfected controls (padj <0.05 and FC ≤ -1 or ≥ 1) [Wald's Test]. Of these 59, 12 EREs were downregulated in PLWH and 47 were upregulated. Colon expression of the ERE loci LTR19_12p13.31 and L1FLnI_1q23.1s showed significant correlations with certain gut immune cell subset frequencies in the colon. Furthermore L1FLnI_1q23.1s showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) of PLWH when compared to uninfected controls suggesting a common mechanism of differential ERE expression in the colon and PBMC. INTERPRETATION: ERE activity has been largely understudied in genomic characterizations of human pathologies. We show that the activity of certain EREs in the colon of PLWH is deregulated, supporting our hypotheses that their underlying activity could function as (bio)markers and potential mediators of pathogenesis in HIV-1 reservoirs. FUNDING: US NIH grants NCI CA260691 (DFN) and NIAID UM1AI164559 (DFN).


Asunto(s)
Retrovirus Endógenos , Infecciones por VIH , VIH-1 , Humanos , Infecciones por VIH/virología , Infecciones por VIH/inmunología , Infecciones por VIH/genética , VIH-1/genética , Retrovirus Endógenos/genética , Masculino , Femenino , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/inmunología , Adulto , Persona de Mediana Edad , Colon/metabolismo , Colon/virología , Colon/patología , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Microbioma Gastrointestinal
3.
Nucleic Acids Res ; 52(9): 5166-5178, 2024 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-38647072

RESUMEN

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.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Ratones Endogámicos C57BL , Animales , Elementos de Nucleótido Esparcido Largo/genética , Ratones , Línea Celular Tumoral , Femenino , Retroelementos/genética , Análisis de Secuencia de ADN/métodos , Humanos , Secuenciación de Nanoporos/métodos , Genoma/genética
4.
Mol Cell ; 84(9): 1637-1650.e10, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38604171

RESUMEN

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Proteínas de Unión a la Región de Fijación a la Matriz , ARN Polimerasa II , Receptores de Estrógenos , Transcripción Genética , Humanos , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Elementos de Nucleótido Esparcido Largo/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Regulación de la Expresión Génica , Unión Proteica , Células HEK293 , Genoma Humano
5.
Mol Genet Genomic Med ; 12(3): e2410, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38433605

RESUMEN

BACKGROUND: Familial hypercholesterolemia (MIM: PS143890) is a genetic disorder characterized by an increase in blood cholesterol. LDLR is one of the genes which their defect contributes to the disorder. Affected individuals may carry a heterozygous variant or homozygous/compound heterozygous variants and those with biallelic pathogenic variants present more severe symptoms. METHOD: We report an Egyptian family with familial hypercholesterolemia. Both the proband and parents have the disorder while a sibling is unaffected. Exome sequencing was performed to identify the causal variant. RESULTS: LINE-1 insertion in exon 7 of LDLR was identified. Both parents have a heterozygous variant while the proband has a homozygous variant. The unaffected sibling did not carry the variant. DISCUSSION: This insertion may contribute to the high prevalence of hypercholesterolemia in Egypt and the finding underscores the importance of implementing mobile element insertion caller in routine bioinformatics pipeline.


Asunto(s)
Hipercolesterolemia , Hiperlipoproteinemia Tipo II , Humanos , Biología Computacional , Egipto , Exones , Hiperlipoproteinemia Tipo II/genética , Elementos de Nucleótido Esparcido Largo
6.
PLoS One ; 19(3): e0298524, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38452152

RESUMEN

The uneven settlement of the surrounding ground surface caused by subway construction is not only complicated but also liable to cause casualties and property damage, so a timely understanding of the ground settlement deformation in the subway excavation and its prediction in real time is of practical significance. Due to the complex nonlinear relationship between subway settlement deformation and numerous influencing factors, as well as the existence of a time lag effect and the influence of various factors in the process, the prediction performance and accuracy of traditional prediction methods can no longer meet industry demands. Therefore, this paper proposes a surface settlement deformation prediction model by combining noise reduction and attention mechanism (AM) with the long short-term memory (LSTM). The complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) and independent component analysis (ICA) methods are used to denoise the input original data and then combined with AM and LSTM for prediction to obtain the CEEMDAN-ICA-AM-LSTM (CIAL) prediction model. Taking the settlement monitoring data of the construction site of Urumqi Rail Transit Line 1 as an example for analysis reveals that the model in this paper has better effectiveness and applicability in the prediction of surface settlement deformation than multiple prediction models. The RMSE, MAE, and MAPE values of the CIAL model are 0.041, 0.033 and 0.384%; R2 is the largest; the prediction effect is the best; the prediction accuracy is the highest; and its reliability is good. The new method is effective for monitoring the safety of surface settlement deformation.


Asunto(s)
Industrias , Vías Férreas , Reproducibilidad de los Resultados , Elementos de Nucleótido Esparcido Largo , Memoria a Largo Plazo
7.
Trends Cancer ; 10(4): 286-288, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38499453

RESUMEN

Subsets of long interspersed nuclear element 1 (LINE-1) retrotransposons can 'retrotranspose' throughout the human genome at a cost to host cell fitness, as observed in some cancers. Pharmacological inhibition of LINE-1 retrotransposition requires a comprehensive understanding of the LINE-1 ORF2p reverse transcriptase. Two recent publications, by Thawani et al. and Baldwin et al., report structures of LINE-1 ORF2p and address long-standing mechanistic gaps regarding LINE-1 retrotransposition. Both studies will be critical to design new specific inhibitors of the LINE-1 ORF2p reverse transcriptase.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Transcripción Reversa , Humanos , Células HeLa , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo
8.
Genes (Basel) ; 15(2)2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38397133

RESUMEN

L1 elements can cause DNA damage and genomic variation via retrotransposition and the generation of endonuclease-dependent DNA breaks. These processes require L1 ORF2p protein that contains an endonuclease domain, which cuts genomic DNA, and a reverse transcriptase domain, which synthesizes cDNA. The complete impact of L1 enzymatic activities on genome stability and cellular function remains understudied, and the spectrum of L1-induced mutations, other than L1 insertions, is mostly unknown. Using an inducible system, we demonstrate that an ORF2p containing functional reverse transcriptase is sufficient to elicit DNA damage response even in the absence of the functional endonuclease. Using a TK/Neo reporter system that captures misrepaired DNA breaks, we demonstrate that L1 expression results in large genomic deletions that lack any signatures of L1 involvement. Using an in vitro cleavage assay, we demonstrate that L1 endonuclease efficiently cuts telomeric repeat sequences. These findings support that L1 could be an unrecognized source of disease-promoting genomic deletions, telomere dysfunction, and an underappreciated source of chronic RT-mediated DNA damage response in mammalian cells. Our findings expand the spectrum of biological processes that can be triggered by functional and nonfunctional L1s, which have impactful evolutionary- and health-relevant consequences.


Asunto(s)
Fenómenos Biológicos , Elementos de Nucleótido Esparcido Largo , Humanos , Animales , Elementos de Nucleótido Esparcido Largo/genética , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Células HeLa , Endonucleasas/genética , Telómero/genética , Telómero/metabolismo , Reparación del ADN/genética , Mamíferos/genética
9.
Cell Genom ; 4(2): 100498, 2024 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-38309261

RESUMEN

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.


Asunto(s)
Metilación de ADN , Retroelementos , Animales , Humanos , Retroelementos/genética , Metilación de ADN/genética , Elementos de Nucleótido Esparcido Largo/genética , Factores de Transcripción/genética , Primates/genética , Epigénesis Genética/genética
10.
Cell Genom ; 4(2): 100504, 2024 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-38359785

RESUMEN

Fully understanding the impact of the human retrotransposon L1 requires that each of ∼500,000 L1 copies be evaluated as a potentially unique genomic entity. In this issue of Cell Genomics, Lanciano et al.1 strive toward this goal, illuminating the reciprocal regulatory influence between individual L1s and their genomic integration sites.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Retroelementos , Humanos , Retroelementos/genética , Elementos de Nucleótido Esparcido Largo/genética , Genómica
11.
Cell Rep ; 43(2): 113749, 2024 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-38329876

RESUMEN

Aberrant long interspersed element 1 (LINE-1 or L1) activity can cause insertional mutagenesis and chromosomal rearrangements and has been detected in several types of cancers. Here, we show that neddylation, a post-translational modification process, is essential for L1 transposition. The antineoplastic drug MLN4924 is an L1 inhibitor that suppresses NEDD8-activating enzyme activity. Neddylation inhibition by MLN4924 selectively impairs ORF2p-mediated L1 reverse transcription and blocks the generation of L1 cDNA. Consistent with these results, MLN4924 treatment suppresses the retrotransposition activity of the non-autonomous retrotransposons short interspersed nuclear element R/variable number of tandem repeat/Alu and Alu, which rely on the reverse transcription activity of L1 ORF2p. The E2 enzyme UBE2M in the neddylation pathway, rather than UBE2F, is required for L1 ORF2p and retrotransposition. Interference with the functions of certain neddylation-dependent Cullin-really interesting new gene E3 ligases disrupts L1 reverse transcription and transposition activity. Our findings provide insights into the regulation of L1 retrotransposition and the identification of therapeutic targets for L1 dysfunctions.


Asunto(s)
Ciclopentanos , Elementos de Nucleótido Esparcido Largo , Pirimidinas , Retroelementos , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Aberraciones Cromosómicas , Proteínas Cullin/genética , Enzimas Ubiquitina-Conjugadoras
12.
Sci Rep ; 14(1): 4322, 2024 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-38383551

RESUMEN

Long interspersed nuclear elements (LINE-1s/L1s) are a group of retrotransposons that can copy themselves within a genome. In humans, it is the most successful transposon in nucleotide content. L1 expression is generally mild in normal human tissues, but the activity has been shown to increase significantly in many cancers. Few studies have examined L1 expression at single-cell resolution, thus it is undetermined whether L1 reactivation occurs solely in malignant cells within tumors. One of the cancer types with frequent L1 activity is high-grade serous ovarian carcinoma (HGSOC). Here, we identified locus-specific L1 expression with 3' single-cell RNA sequencing in pre- and post-chemotherapy HGSOC sample pairs from 11 patients, and in fallopian tube samples from five healthy women. Although L1 expression quantification with the chosen technique was challenging due to the repetitive nature of the element, we found evidence of L1 expression primarily in cancer cells, but also in other cell types, e.g. cancer-associated fibroblasts. The expression levels were similar in samples taken before and after neoadjuvant chemotherapy, indicating that L1 transcriptional activity was unaffected by clinical platinum-taxane treatment. Furthermore, L1 activity was negatively associated with the expression of MYC target genes, a finding that supports earlier literature of MYC being an L1 suppressor.


Asunto(s)
Neoplasias Ováricas , Humanos , Femenino , Neoplasias Ováricas/patología , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Trompas Uterinas/metabolismo
13.
Cell Rep ; 43(2): 113774, 2024 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-38349791

RESUMEN

Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.


Asunto(s)
Inestabilidad Genómica , Células-Madre Neurales , Humanos , Animales , Ratones , Diferenciación Celular , Elementos de Nucleótido Esparcido Largo
14.
Nucleic Acids Res ; 52(6): 3327-3345, 2024 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-38197223

RESUMEN

LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3' ends' dynamics and the interplay between L1 3' and 5' ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5'→3' exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3' poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3' ends demonstrating the important role of L1 3' end dynamics in L1 biology.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , ARN Mensajero , Humanos , Células HeLa , Retroelementos/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
15.
EMBO J ; 43(1): 112-131, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38177314

RESUMEN

Transposable elements have created the majority of the sequence in many genomes. In mammals, LINE-1 retrotransposons have been expanding for more than 100 million years as distinct, consecutive lineages; however, the drivers of this recurrent lineage emergence and disappearance are unknown. Most human genome assemblies provide a record of this ancient evolution, but fail to resolve ongoing LINE-1 retrotranspositions. Utilizing the human CHM1 long-read-based haploid assembly, we identified and cloned all full-length, intact LINE-1s, and found 29 LINE-1s with measurable in vitro retrotransposition activity. Among individuals, these LINE-1s varied in their presence, their allelic sequences, and their activity. We found that recently retrotransposed LINE-1s tend to be active in vitro and polymorphic in the population relative to more ancient LINE-1s. However, some rare allelic forms of old LINE-1s retain activity, suggesting older lineages can persist longer than expected. Finally, in LINE-1s with in vitro activity and in vivo fitness, we identified mutations that may have increased replication in ancient genomes and may prove promising candidates for mechanistic investigations of the drivers of LINE-1 evolution and which LINE-1 sequences contribute to human disease.


Asunto(s)
Genoma Humano , Elementos de Nucleótido Esparcido Largo , Animales , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos , Mamíferos/genética , Mutación , Evolución Molecular
16.
Cancer Sci ; 115(1): 247-256, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38013627

RESUMEN

The long interspersed nuclear element-1 (LINE-1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE-1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE-1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE-1 hypomethylation, retrotransposition, and tumor-specific insertion in upper GI cancers remain unknown. We used bisulfite-pyrosequencing and quantitative real-time PCR to verify LINE-1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE-1 retrotransposition profile with an originally developed L1Hs-seq. In tumor samples, LINE-1 methylation levels were significantly lower than non-tumor controls, while LINE-1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE-1 methylation level and copy number in tumor tissues, with lower LINE-1 methylation levels corresponding to higher LINE-1 copy numbers. Of particular importance is that somatic LINE-1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE-1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor-suppressive genes. LINE-1 hypomethylation in upper GI cancers is related to increased LINE-1 retrotransposition and tumor-specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor-suppressive genes.


Asunto(s)
Neoplasias Gastrointestinales , Neoplasias Gástricas , Humanos , Metilación de ADN/genética , Neoplasias Gastrointestinales/genética , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias Gástricas/genética , Esófago
17.
Nature ; 626(7997): 186-193, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38096901

RESUMEN

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.


Asunto(s)
ADN Complementario , Elementos de Nucleótido Esparcido Largo , ARN , Retroelementos , Transcripción Reversa , Humanos , Microscopía por Crioelectrón , ADN Complementario/biosíntesis , ADN Complementario/genética , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , ARN/química , ARN/genética , ARN/metabolismo , Dominio Catalítico , Endonucleasas/química , Endonucleasas/metabolismo , Endonucleasas/ultraestructura , Terapia Genética , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/metabolismo , ADN Polimerasa Dirigida por ARN/ultraestructura , ADN de Cadena Simple/metabolismo , Roturas del ADN
18.
Nature ; 626(7997): 194-206, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38096902

RESUMEN

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.


Asunto(s)
Endonucleasas , Elementos de Nucleótido Esparcido Largo , ADN Polimerasa Dirigida por ARN , Transcripción Reversa , Humanos , Microscopía por Crioelectrón , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , ARN/genética , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Cristalografía por Rayos X , ADN/biosíntesis , ADN/genética , Inmunidad Innata , Interferones/biosíntesis
19.
Nat Commun ; 14(1): 8217, 2023 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-38086852

RESUMEN

Cyclic GMP-AMP synthase (cGAS), initially identified as a cytosolic DNA sensor, detects DNA fragments to trigger an innate immune response. Recently, accumulating evidence reveals the presence of cGAS within the nucleus. However, the biological functions of nuclear cGAS are not fully understood. Here, we demonstrate that nuclear cGAS represses LINE-1 (L1) retrotransposition to preserve genome integrity in human cells. Mechanistically, the E3 ligase TRIM41 interacts with and ubiquitinates ORF2p to influence its stability, and cGAS enhances the association of ORF2p with TRIM41, thereby promoting TRIM41-mediated ORF2p degradation and the suppression of L1 retrotransposition. In response to DNA damage, cGAS is phosphorylated at serine residues 120 and 305 by CHK2, which promotes cGAS-TRIM41 association, facilitating TRIM41-mediated ORF2p degradation. Moreover, we show that nuclear cGAS mediates the repression of L1 retrotransposition in senescent cells induced by DNA damage agents. We also identify several cancer-associated cGAS mutations that abolish the suppressive effect on L1 retrotransposition by disrupting the CHK2-cGAS-TRIM41-ORF2p regulatory axis. Together, these findings indicate that nuclear cGAS exhibits an inhibitory function in L1 retrotransposition which could provide avenues for future interventions in both aging and tumorigenesis.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Nucleotidiltransferasas , Humanos , ADN/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
20.
Genes Dev ; 37(21-24): 948-967, 2023 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-38092519

RESUMEN

Long interspersed element 1 (LINE-1) is the only protein-coding transposon that is active in humans. LINE-1 propagates in the genome using RNA intermediates via retrotransposition. This activity has resulted in LINE-1 sequences occupying approximately one-fifth of our genome. Although most copies of LINE-1 are immobile, ∼100 copies are retrotransposition-competent. Retrotransposition is normally limited via epigenetic silencing, DNA repair, and other host defense mechanisms. In contrast, LINE-1 overexpression and retrotransposition are hallmarks of cancers. Here, we review mechanisms of LINE-1 regulation and how LINE-1 may promote genetic heterogeneity in tumors. Finally, we discuss therapeutic strategies to exploit LINE-1 biology in cancers.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Neoplasias , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Neoplasias/genética , Neoplasias/terapia , ARN , Proteínas/genética , Epigénesis Genética
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