RESUMEN
Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism activated in ~10-15% of cancers, characterized by telomeric damage. Telomeric damage-induced long non-coding RNAs (dilncRNAs) are transcribed at dysfunctional telomeres and contribute to telomeric DNA damage response (DDR) activation and repair. Here we observed that telomeric dilncRNAs are preferentially elevated in ALT cells. Inhibition of C-rich (teloC) dilncRNAs with antisense oligonucleotides leads to DNA replication stress responses, increased genomic instability, and apoptosis induction selectively in ALT cells. Cell death is dependent on DNA replication and is increased by DNA replication stress. Mechanistically, teloC dilncRNA inhibition reduces RAD51 and 53BP1 recruitment to telomeres, boosts the engagement of BIR machinery, and increases C-circles and telomeric sister chromatid exchanges, without increasing telomeric non-S phase synthesis. These results indicate that teloC dilncRNA is necessary for a coordinated recruitment of DDR factors to ALT telomeres and it is essential for ALT cancer cells survival.
Asunto(s)
Telomerasa , Homeostasis del Telómero , Homeostasis del Telómero/genética , Replicación del ADN , ARN , Supervivencia Celular/genética , Telómero/genética , Telómero/metabolismo , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Ageing organisms accumulate senescent cells that are thought to contribute to body dysfunction. Telomere shortening and damage are recognized causes of cellular senescence and ageing. Several human conditions associated with normal ageing are precipitated by accelerated telomere dysfunction. Here, we systematize a large body of evidence and propose a coherent perspective to recognize the broad contribution of telomeric dysfunction to human pathologies.
Asunto(s)
Envejecimiento/metabolismo , Senescencia Celular , Enfermedades no Transmisibles , Homeostasis del Telómero , Acortamiento del Telómero , Telómero/metabolismo , Factores de Edad , Envejecimiento/genética , Envejecimiento/patología , Animales , Daño del ADN , Humanos , Telómero/genética , Telómero/patologíaRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), known to be more common in the elderly, who also show more severe symptoms and are at higher risk of hospitalization and death. Here, we show that the expression of the angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 cell receptor, increases during aging in mouse and human lungs. ACE2 expression increases upon telomere shortening or dysfunction in both cultured mammalian cells and in vivo in mice. This increase is controlled at the transcriptional level, and Ace2 promoter activity is DNA damage response (DDR)-dependent. Both pharmacological global DDR inhibition of ATM kinase activity and selective telomeric DDR inhibition by the use of antisense oligonucleotides prevent Ace2 upregulation following telomere damage in cultured cells and in mice. We propose that during aging telomere dysfunction due to telomeric shortening or damage triggers DDR activation and this causes the upregulation of ACE2, the SARS-CoV-2 cell receptor, thus contributing to make the elderly more susceptible to the infection.
Asunto(s)
Envejecimiento , Enzima Convertidora de Angiotensina 2/genética , COVID-19 , Daño del ADN , Telómero , Anciano , Envejecimiento/genética , Animales , Humanos , Ratones , SARS-CoV-2 , Telómero/genéticaRESUMEN
Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2T188A) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease.
Asunto(s)
Aterosclerosis/patología , Senescencia Celular , Inflamación/patología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Neointima/patología , Telómero/patología , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Proliferación Celular , Células Cultivadas , Daño del ADN , Modelos Animales de Enfermedad , Humanos , Inflamación/etiología , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/fisiología , Proteínas Musculares/fisiología , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , Neointima/etiología , Neointima/metabolismo , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disorder characterized by premature aging features. Cells from HGPS patients express progerin, a truncated form of Lamin A, which perturbs cellular homeostasis leading to nuclear shape alterations, genome instability, heterochromatin loss, telomere dysfunction and premature entry into cellular senescence. Recently, we reported that telomere dysfunction induces the transcription of telomeric non-coding RNAs (tncRNAs) which control the DNA damage response (DDR) at dysfunctional telomeres. Here we show that progerin-induced telomere dysfunction induces the transcription of tncRNAs. Their functional inhibition by sequence-specific telomeric antisense oligonucleotides (tASOs) prevents full DDR activation and premature cellular senescence in various HGPS cell systems, including HGPS patient fibroblasts. We also show in vivo that tASO treatment significantly enhances skin homeostasis and lifespan in a transgenic HGPS mouse model. In summary, our results demonstrate an important role for telomeric DDR activation in HGPS progeroid detrimental phenotypes in vitro and in vivo.
Asunto(s)
Daño del ADN , Progeria/patología , Telómero/metabolismo , Animales , Línea Celular , Proliferación Celular , Senescencia Celular , Reparación del ADN , Modelos Animales de Enfermedad , Homeostasis , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Ratones , Mutación/genética , Oligonucleótidos Antisentido/metabolismo , Fenotipo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Piel/patologíaRESUMEN
Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.
Asunto(s)
Daño del ADN , Reparación del ADN , ARN no Traducido , Ribonucleasa Pancreática/metabolismo , Animales , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , Técnicas Genéticas , Células HeLa , Humanos , Ratones , Células 3T3 NIH , ARN/análisis , ARN/genética , ARN/metabolismo , ARN no Traducido/genética , ARN no Traducido/fisiologíaRESUMEN
Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.
Asunto(s)
Inestabilidad Genómica , ARN no Traducido/genética , Roturas del ADN de Doble Cadena , Daño del ADN , Regulación de la Expresión Génica , Humanos , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo , Transcripción GenéticaRESUMEN
Identification of important, functional small RNA (sRNA) species is currently hampered by the lack of reliable and sensitive methods to isolate and characterize them. We have developed a method, termed target-enrichment of sRNAs (TEsR), that enables targeted sequencing of rare sRNAs and diverse precursor and mature forms of sRNAs not detectable by current standard sRNA sequencing methods. It is based on the amplification of full-length sRNA molecules, production of biotinylated RNA probes, hybridization to one or multiple targeted RNAs, removal of nontargeted sRNAs and sequencing. By this approach, target sRNAs can be enriched by a factor of 500-30,000 while maintaining strand specificity. TEsR enriches for sRNAs irrespective of length or different molecular features, such as the presence or absence of a 5' cap or of secondary structures or abundance levels. Moreover, TEsR allows the detection of the complete sequence (including sequence variants, and 5' and 3' ends) of precursors, as well as intermediate and mature forms, in a quantitative manner. A well-trained molecular biologist can complete the TEsR procedure, from RNA extraction to sequencing library preparation, within 4-6 d.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/aislamiento & purificación , Análisis de Secuencia de ARN/métodosRESUMEN
The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.
Asunto(s)
Daño del ADN , ARN/metabolismo , Telómero/metabolismo , Animales , Ratones Endogámicos C57BL , Ratones Noqueados , Oligonucleótidos Antisentido/farmacología , ARN/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/genética , Ribonucleasa III/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.
Asunto(s)
Senescencia Celular , Daño del ADN , Reparación del ADN , Fibroblastos/citología , Anciano , Ciclo Celular , Línea Celular , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Fibroblastos/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Radiación Ionizante , Transducción de Señal , Telómero/ultraestructura , beta-Galactosidasa/metabolismoRESUMEN
The DNA damage response (DDR) orchestrates DNA repair and halts cell cycle. If damage is not resolved, cells can enter into an irreversible state of proliferative arrest called cellular senescence. Organismal ageing in mammals is associated with accumulation of markers of cellular senescence and DDR persistence at telomeres. Since the vast majority of the cells in mammals are non-proliferating, how do they age? Are telomeres involved? Also oncogene activation causes cellular senescence due to altered DNA replication and DDR activation in particular at the telomeres. Is there a common mechanism shared among apparently distinct types of cellular senescence? And what is the role of telomeric DNA damage?
Asunto(s)
Envejecimiento/genética , Senescencia Celular/genética , Daño del ADN , Reparación del ADN/genética , Transducción de Señal/genética , Animales , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Modelos Genéticos , Telómero/genéticaRESUMEN
Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron-exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3'UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3'UTR for mutations in a large series of BRCA-mutation negative, population and clinic-based breast cancer cases, and controls. Fifteen novel BRCA1 3'UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.* 528G>C, c.* 718A>G, and c.* 1271T>C and four found in controls, c.* 309T>C, c.* 379G>A, c.* 823C>T, and c.* 264C>T, reduced 3'UTR activity (P < 0.02), whereas two variants found in cases, c.* 291C>T and c.* 1139G>T, increased 3'UTR activity (P < 0.01). Three case variants, c.* 718A>G, c.* 800T>C, and c.* 1340_1342delTGT, were predicted to create new miRNA binding sites and c.* 1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3'UTR reporter activity when coexpressed with the predicted targeting miRNA, miR-103. This is the most comprehensive identification and analysis of BRCA1 3'UTR variants published to date.
Asunto(s)
Regiones no Traducidas 3' , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Mutación de Línea Germinal , MicroARNs/genética , Adulto , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Estudios de Casos y Controles , Línea Celular Tumoral , Secuencia Conservada , Análisis Mutacional de ADN , Proteínas ELAV , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Humanos , Conformación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Unión Proteica , Interferencia de ARN , ARN Mensajero/genéticaRESUMEN
The DNA-damage response (DDR) arrests cell-cycle progression until damage is removed. DNA-damage-induced cellular senescence is associated with persistent DDR. The molecular bases that distinguish transient from persistent DDR are unknown. Here we show that a large fraction of exogenously induced persistent DDR markers is associated with telomeric DNA in cultured cells and mammalian tissues. In yeast, a chromosomal DNA double-strand break next to a telomeric sequence resists repair and impairs DNA ligase 4 recruitment. In mammalian cells, ectopic localization of telomeric factor TRF2 next to a double-strand break induces persistent DNA damage and DDR. Linear, but not circular, telomeric DNA or scrambled DNA induces a prolonged checkpoint in normal cells. In terminally differentiated tissues of old primates, DDR markers accumulate at telomeres that are not critically short. We propose that linear genomes are not uniformly reparable and that telomeric DNA tracts, if damaged, are irreparable and trigger persistent DDR and cellular senescence.