RESUMEN
TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43-dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding-dependent inter-RRM interactions are crucial for TDP-43 function.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Empalme del ARN/fisiología , Proteínas de Unión al ARN/fisiología , Secuencia de Aminoácidos , Composición de Base , Sitios de Unión , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts. Our data reinforce the hypothesis that progressive chromatin destabilization may lead to the loss of epigenetic information and impaired cellular function associated with chronic DNA damage upon drug-evoked senescence. We propose that changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging. In addition, we also outline the method that allows for quantitative and unbiased measurement of these changes.
Asunto(s)
Senescencia Celular/genética , Daño del ADN/genética , Histonas/genética , Transducción de Señal/genética , Secuencia de Aminoácidos , Antibióticos Antineoplásicos , Bleomicina , Western Blotting , Senescencia Celular/efectos de los fármacos , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Inmunohistoquímica , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA-binding proteins pathogenetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but it is not known if they regulate the same transcripts. We addressed this question using crosslinking and immunoprecipitation (iCLIP) in mouse brain, which showed that FUS binds along the whole length of the nascent RNA with limited sequence specificity to GGU and related motifs. A saw-tooth binding pattern in long genes demonstrated that FUS remains bound to pre-mRNAs until splicing is completed. Analysis of FUS(-/-) brain demonstrated a role for FUS in alternative splicing, with increased crosslinking of FUS in introns around the repressed exons. We did not observe a significant overlap in the RNA binding sites or the exons regulated by FUS and TDP-43. Nevertheless, we found that both proteins regulate genes that function in neuronal development.
Asunto(s)
Empalme Alternativo , Encéfalo/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exones , Regulación de la Expresión Génica , Orden Génico , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Unión Proteica , Isoformas de ARN , Proteína FUS de Unión a ARN/genéticaRESUMEN
Recent analyses suggest that transposable element-derived transcripts are processed to yield a variety of small RNA species that play critical functional roles in gene regulation and chromatin organization as well as genome stability and maintenance. Here we report a mass spectrometry analysis of an RNA-affinity complex isolation using a piRNA homologous sequence derived from Alu retrotransposal RNA. Our data point to potential roles for piALU RNAs in DNA repair, cell cycle and chromatin regulations.
RESUMEN
Emerging evidence is shedding light on a large and complex network of epigenetic modifications at play in human stem cells. This "epigenetic landscape" governs the fine-tuning and precision of gene expression programs that define the molecular basis of stem cell pluripotency, differentiation and reprogramming. This review will focus on recent progress in our understanding of the processes that govern this landscape in stem cells, such as histone modification, DNA methylation, alterations of chromatin structure due to chromatin remodeling and non-coding RNA activity. Further investigation into stem cell epigenetics promises to provide novel advances in the diagnosis and treatment of a wide array of human diseases.
Asunto(s)
Diferenciación Celular/genética , Epigénesis Genética/genética , Células Madre Pluripotentes/metabolismo , Animales , Cromatina/química , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Metilación de ADN , Histonas/metabolismo , Humanos , Células Madre Pluripotentes/citología , Procesamiento Proteico-Postraduccional , ARN no Traducido/metabolismoRESUMEN
Adult stem cells have taken center stage in current research related to regenerative medicine and pharmacogenomic studies seeking new therapeutic interventions. As we learn more about these cells, it is becoming apparent that the next big leap in our understanding of adult stem cell biology and adult stem cell aging will depend on the integration of approaches from various disciplines. Major advances and technological breakthroughs at the crossroad of fields such as biomaterials, genomics, epigenomics, and proteomics will enable the design of better tools to model human diseases, and warrant safe usage of adult stem cells in the clinic.
Asunto(s)
Células Madre Adultas/fisiología , Envejecimiento/fisiología , Modelos Biológicos , Medicina Regenerativa/métodos , Investigación/tendencias , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Humanos , Medicina Regenerativa/tendenciasRESUMEN
Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of individuals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)-dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)-dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration.
Asunto(s)
Envejecimiento , Empalme Alternativo , Enfermedad de Alzheimer/genética , Degeneración Lobar Frontotemporal/genética , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/genética , Regulación hacia Abajo , Exones , Perfilación de la Expresión Génica , Humanos , Canales Iónicos/genética , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Antígeno Ventral Neuro-Oncológico , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Análisis de Componente Principal , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transmisión Sináptica/genética , Lóbulo Temporal/metabolismo , Transcripción Genética , Adulto JovenRESUMEN
TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.
Asunto(s)
Empalme Alternativo/genética , Química Encefálica/genética , Proteínas de Unión al ADN/genética , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/genética , Humanos , Isoformas de Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN no Traducido/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3' UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3' UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Retroalimentación Fisiológica/fisiología , Regulación de la Expresión Génica/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/metabolismo , Secuencia de Bases , Northern Blotting , Línea Celular , Proteínas de Unión al ADN/genética , Biblioteca de Genes , Humanos , Immunoblotting , Inmunoprecipitación , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Interferencia de ARN , Análisis de Secuencia de ADNRESUMEN
UV cross-linking and immunoprecipitation assay (CLIP) can identify direct interaction sites between RNA-binding proteins and RNAs in vivo, and has been used to study several proteins in tissues and cell cultures. The main challenge of the method is to specifically amplify the low amount of isolated RNA. The current protocol is optimised for efficient RNA purification and ligation of barcoded RNA adapters. High-throughput sequencing of the multiplexed cDNA library allows for a comprehensive coverage of the target sequences.