RESUMEN
DNA hydroxymethylation (5hmC), the most abundant oxidative derivative of DNA methylation, is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in age-related diseases, its functional role in aging remains unknown. Here, using mouse liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and restricts the magnitude of gene expression changes with age. Mechanistically, 5hmC decreases the binding of splicing associated factors and correlates with age-related alternative splicing events. We found that various age-related contexts, such as prolonged quiescence and senescence, drive the accumulation of 5hmC with age. We provide evidence that this age-related transcriptionally restrictive function is conserved in mouse and human tissues. Our findings reveal that 5hmC regulates tissue-specific function and may play a role in longevity.
Asunto(s)
5-Metilcitosina , Envejecimiento , Cerebelo , Metilación de ADN , Hígado , Animales , Envejecimiento/genética , Envejecimiento/metabolismo , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Hígado/metabolismo , Ratones , Humanos , Cerebelo/metabolismo , Ratones Endogámicos C57BL , Longevidad/genética , Masculino , Empalme Alternativo , Transcripción Genética , Femenino , Regulación de la Expresión GénicaRESUMEN
Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/genética , Péptidos , ProteómicaRESUMEN
Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on removal of the poly(A) tail. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome. Inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 fully rescues RNA length and suppresses stress-induced decay. Our findings reveal RNA decay as a key determinant of RNA metabolism upon cellular stress and dependent on stress-granule formation.
RESUMEN
Quantification of the dynamics of RNA metabolism is essential for understanding gene regulation in health and disease. Existing methods rely on metabolic labeling of nascent RNAs and physical separation or inference of labeling through PCR-generated mutations, followed by short-read sequencing. However, these methods are limited in their ability to identify transient decay intermediates or co-analyze RNA decay with cis-regulatory elements of RNA stability such as poly(A) tail length and modification status, at single molecule resolution. Here we use 5-ethynyl uridine (5EU) to label nascent RNA followed by direct RNA sequencing with nanopores. We developed RNAkinet, a deep convolutional and recurrent neural network that processes the electrical signal produced by nanopore sequencing to identify 5EU-labeled nascent RNA molecules. RNAkinet demonstrates generalizability to distinct cell types and organisms and reproducibly quantifies RNA kinetic parameters allowing the combined interrogation of RNA metabolism and cis-acting RNA regulatory elements.
RESUMEN
Senescence is a state of indefinite cell cycle arrest associated with aging, cancer, and age-related diseases. Here, using label-based mass spectrometry, ribosome profiling and nanopore direct RNA sequencing, we explore the coordinated interaction of translational and transcriptional programs of human cellular senescence. We find that translational deregulation and a corresponding maladaptive integrated stress response (ISR) is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2α phosphorylation, typical of ISR activation, but translationally repress production of the stress response transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames. Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. Furthermore, absent a response, stress augments the senescence secretory phenotype, thus intensifying a proinflammatory state that exacerbates disease. Our results reveal a novel mechanism that senescent cells exploit to evade an adaptive stress response and remain viable.
RESUMEN
DNA hydroxymethylation (5hmC) is the most abundant oxidative derivative of DNA methylation (5mC) and is typically enriched at enhancers and gene bodies of transcriptionally active and tissue-specific genes. Although aberrant genomic 5hmC has been implicated in many age-related diseases, the functional role of the modification in aging remains largely unknown. Here, we report that 5hmC is stably enriched in multiple aged organs. Using the liver and cerebellum as model organs, we show that 5hmC accumulates in gene bodies associated with tissue-specific function and thereby restricts the magnitude of gene expression changes during aging. Mechanistically, we found that 5hmC decreases binding affinity of splicing factors compared to unmodified cytosine and 5mC, and is correlated with age-related alternative splicing events, suggesting RNA splicing as a potential mediator of 5hmC's transcriptionally restrictive function. Furthermore, we show that various age-related contexts, such as prolonged quiescence and senescence, are partially responsible for driving the accumulation of 5hmC with age. We provide evidence that this age-related function is conserved in mouse and human tissues, and further show that the modification is altered by regimens known to modulate lifespan. Our findings reveal that 5hmC is a regulator of tissue-specific function and may play a role in regulating longevity.
RESUMEN
Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.
RESUMEN
A defining feature of embryonic stem cells (ESCs) is the ability to differentiate into all three germ layers. Pluripotency is maintained in part by a unique transcription network that maintains expression of pluripotency-specific transcription factors and represses developmental genes. While the mechanisms that establish this transcription network are well studied, little is known of the posttranscriptional surveillance pathways that degrade differentiation-related RNAs. We report that the surveillance pathway mediated by the RNA exosome nuclease complex represses ESC differentiation. Depletion of the exosome expedites differentiation of human ESCs into all three germ layers. LINE-1 retrotransposons and specific miRNAs, lncRNAs, and mRNAs that encode developmental regulators or affect their expression are all bound by the exosome and increase in level upon exosome depletion. The exosome restrains differentiation in part by degrading transcripts encoding FOXH1, a transcription factor crucial for mesendoderm formation. Our studies establish the exosome as a regulator of human ESC differentiation and reveal the importance of RNA decay in maintaining pluripotency.
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Diferenciación Celular , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Reactivos de Enlaces Cruzados/química , Endodermo/embriología , Endodermo/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Mesodermo/embriología , Mesodermo/metabolismo , MicroARNs/genética , Fenotipo , ARN/aislamiento & purificación , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transcripción Genética , TransgenesRESUMEN
Numerous surveillance pathways sculpt eukaryotic transcriptomes by degrading unneeded, defective, and potentially harmful noncoding RNAs (ncRNAs). Because aberrant and excess ncRNAs are largely degraded by exoribonucleases, a key characteristic of these RNAs is an accessible, protein-free 5' or 3' end. Most exoribonucleases function with cofactors that recognize ncRNAs with accessible 5' or 3' ends and/or increase the availability of these ends. Noncoding RNA surveillance pathways were first described in budding yeast, and there are now high-resolution structures of many components of the yeast pathways and significant mechanistic understanding as to how they function. Studies in human cells are revealing the ways in which these pathways both resemble and differ from their yeast counterparts, and are also uncovering numerous pathways that lack equivalents in budding yeast. In this review, we describe both the well-studied pathways uncovered in yeast and the new concepts that are emerging from studies in mammalian cells. We also discuss the ways in which surveillance pathways compete with chaperone proteins that transiently protect nascent ncRNA ends from exoribonucleases, with partner proteins that sequester these ends within RNPs, and with end modification pathways that protect the ends of some ncRNAs from nucleases.
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ADN de Hongos/genética , ARN no Traducido/genética , Saccharomyces cerevisiae/genética , Animales , Exosomas/metabolismo , HumanosRESUMEN
Non-coding RNAs (ncRNAs) called Y RNAs are abundant components of both animal cells and a variety of bacteria. In all species examined, these ~100 nt RNAs are bound to the Ro 60 kDa (Ro60) autoantigen, a ring-shaped protein that also binds misfolded ncRNAs in some vertebrate nuclei. Although the function of Ro60 RNPs has been mysterious, we recently reported that a bacterial Y RNA tethers Ro60 to the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNPase) to form RYPER (Ro60/Y RNA/PNPase Exoribonuclease RNP), a new RNA degradation machine. PNPase is a homotrimeric ring that degrades single-stranded RNA, and Y RNA-mediated tethering of Ro60 increases the effectiveness of PNPase in degrading structured RNAs. Single particle electron microscopy of RYPER suggests that RNA threads through the Ro60 ring into the PNPase cavity. Further studies indicate that Y RNAs may also act as gates to regulate entry of RNA substrates into the Ro60 channel. These findings reveal novel functions for Y RNAs and raise questions about how the bacterial findings relate to the roles of these ncRNAs in animal cells. Here we review the literature on Y RNAs, highlighting their close relationship with Ro60 proteins and the hypothesis that these ncRNAs function generally to tether Ro60 rings to diverse RNA-binding proteins.
Asunto(s)
ARN no Traducido/metabolismo , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN no Traducido/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
BACKGROUND: MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. RESULTS: Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. CONCLUSIONS: These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections.
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Loop-loop interactions mediate the recognition between RNA hairpins leading to the formation of so-called kissing complexes. Both the size and the sequence of the loop are critical for ensuring stable interaction. Using in vitro selection we have characterized a few loop sequences that lead to the formation of highly stable kissing complexes. These sequences constitute targets of interest for the rational design of RNA stem loop ligands. Such an appropriate target sequence was identified in a sub-domain of the Internal Ribosomal Entry Site (IRES) of the Hepatitis C Virus (HCV) mRNA. We synthesized chemically-modified RNA hairpins and demonstrated that they specifically reduced the expression of a HCV IRES driven reporter gene in cultured cells.