RESUMEN
Nonsense-mediated decay (NMD) is a translation-dependent RNA quality control mechanism that occurs in the cytoplasm. However, it is unknown how NMD regulates the stability of RNAs translated at the endoplasmic reticulum (ER). Here, we identify a localized NMD pathway dedicated to ER-translated mRNAs. We previously identified NBAS, a component of the Syntaxin 18 complex involved in Golgi-to-ER trafficking, as a novel NMD factor. Furthermore, we show that NBAS fulfills an independent function in NMD. This ER-NMD pathway requires the interaction of NBAS with the core NMD factor UPF1, which is partially localized at the ER in the proximity of the translocon. NBAS and UPF1 coregulate the stability of ER-associated transcripts, in particular those associated with the cellular stress response. We propose a model where NBAS recruits UPF1 to the membrane of the ER and activates an ER-dedicated NMD pathway, thus providing an ER-protective function by ensuring quality control of ER-translated mRNAs.
Asunto(s)
Retículo Endoplásmico/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Retículo Endoplásmico/enzimología , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Biosíntesis de Proteínas , ARN Helicasas/metabolismoRESUMEN
The DExD/H-box RNA helicase DHX34 is a nonsense-mediated decay (NMD) factor that together with core NMD factors coregulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using cross-linking immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial myelodysplasia (MDS)/acute myeloid leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggest that alterations in the levels and/or activity of DHX34 could contribute to human disease.
Asunto(s)
Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Empalme Alternativo , Animales , Humanos , Leucemia Mieloide Aguda/genética , Mamíferos/genética , Síndromes Mielodisplásicos/genética , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas/genética , ARN Helicasas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/genéticaRESUMEN
The Nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs) but also regulates the abundance of a large number of cellular RNAs. The central role of NMD in the control of gene expression requires the existence of buffering mechanisms that tightly regulate the magnitude of this pathway. Here, we will focus on the mechanism of NMD with an emphasis on the role of RNA helicases in the transition from NMD complexes that recognize a PTC to those that promote mRNA decay. We will also review recent strategies aimed at uncovering novel trans-acting factors and their functional role in the NMD pathway. Finally, we will describe recent progress in the study of the physiological role of the NMD response.
Asunto(s)
Codón sin Sentido/genética , Regulación de la Expresión Génica/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Estabilidad del ARN/genética , Humanos , Redes y Vías Metabólicas/genética , ARN Helicasas/genética , ARN Mensajero/genéticaRESUMEN
Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process.
Asunto(s)
Caenorhabditis elegans/genética , Proteínas Portadoras/metabolismo , Drosophila melanogaster/genética , Proteínas de Unión al GTP/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido/fisiología , Proteínas Nucleares/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Embrión no Mamífero , Evolución Molecular , Proteínas de Unión al GTP/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons but also regulates the abundance of cellular RNAs. We sought to identify transcripts that are regulated by two novel NMD factors, DHX34 and neuroblastoma amplified sequence (NBAS), which were identified in a genome-wide RNA interference screen in Caenorhabditis elegans and later shown to mediate NMD in vertebrates. We performed microarray expression profile analysis in human cells, zebrafish embryos and C. elegans that were individually depleted of these factors. Our analysis revealed that a significant proportion of genes are co-regulated by DHX34, NBAS and core NMD factors in these three organisms. Further analysis indicates that NMD modulates cellular stress response pathways and membrane trafficking across species. Interestingly, transcripts encoding different NMD factors were sensitive to DHX34 and NBAS depletion, suggesting that these factors participate in a conserved NMD negative feedback regulatory loop, as was recently described for core NMD factors. In summary, we find that DHX34 and NBAS act in concert with core NMD factors to co-regulate a large number of endogenous RNA targets. Furthermore, the conservation of a mechanism to tightly control NMD homeostasis across different species highlights the importance of the NMD response in the control of gene expression.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Evolución Molecular , Perfilación de la Expresión Génica , Células HeLa , Homeostasis , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , ARN Helicasas/antagonistas & inhibidores , ARN Mensajero/metabolismo , Transactivadores/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
Telomeres can fold into t-loops that may result from the invasion of the 3' overhang into duplex DNA. Their formation is facilitated in vitro by the telomeric protein TRF2, but very little is known regarding the mechanisms involved. Here we reveal that TRF2 generates positive supercoiling and condenses DNA. Using a variety of TRF2 mutants, we demonstrate a strong correlation between this topological activity and the ability to stimulate strand invasion. We also report that these properties require the combination of the TRF-homology (TRFH) domain of TRF2 with either its N- or C-terminal DNA-binding domains. We propose that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, can induce untwisting of the neighboring DNA, thereby favoring strand invasion. Implications of this topological model in t-loop formation and telomere homeostasis are discussed.
Asunto(s)
ADN/química , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/química , ADN Superhelicoidal/química , Humanos , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico , Proteína 1 de Unión a Repeticiones Teloméricas/químicaRESUMEN
Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , ADN Helicasas/metabolismo , ARN Helicasas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Proteínas Portadoras/genética , Clonación Molecular , ADN Helicasas/genética , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Humanos , ARN Helicasas/genéticaRESUMEN
The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.
Asunto(s)
Mutación de Línea Germinal , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Desaminasa/genética , Dineínas Axonemales/genética , Estudios de Cohortes , Humanos , Degradación de ARNm Mediada por Codón sin Sentido , Linaje , Perforina/genética , Glicoproteínas de Membrana Plaquetaria/genética , ARN Helicasas/genética , Receptores de Interleucina-17/genética , Proteínas de Transporte Vesicular/genética , Secuenciación del ExomaRESUMEN
The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast.
Asunto(s)
Codón sin Sentido , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Proteínas Portadoras/química , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Biosíntesis de Proteínas , ARN Helicasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Sirolimus/farmacología , Telomerasa/genéticaRESUMEN
MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast and gastric cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlight that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis.
Asunto(s)
Neoplasias de la Mama/genética , MicroARNs/genética , Procesamiento Postranscripcional del ARN/genética , Neoplasias de la Mama/metabolismo , Femenino , Variación Genética , Células HEK293 , Humanos , Técnicas In Vitro , Células MCF-7 , MicroARNs/metabolismo , Modelos Genéticos , Conformación de Ácido Nucleico , ARN , Ribonucleasa III/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMEN
Nonsense-mediated decay (NMD) is a messenger RNA quality-control pathway triggered by SMG1-mediated phosphorylation of the NMD factor UPF1. In recent times, the RNA helicase DHX34 was found to promote mRNP remodelling, leading to activation of NMD. Here we demonstrate the mechanism by which DHX34 functions in concert with SMG1. DHX34 comprises two distinct structural units, a core that binds UPF1 and a protruding carboxy-terminal domain (CTD) that binds the SMG1 kinase, as shown using truncated forms of DHX34 and electron microscopy of the SMG1-DHX34 complex. Truncation of the DHX34 CTD does not affect binding to UPF1; however, it compromises DHX34 binding to SMG1 to affect UPF1 phosphorylation and hence abrogate NMD. Altogether, these data suggest the existence of a complex comprising SMG1, UPF1 and DHX34, with DHX34 functioning as a scaffold for UPF1 and SMG1. This complex promotes UPF1 phosphorylation leading to functional NMD.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Helicasas/metabolismo , Transactivadores/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Técnicas In Vitro , Microscopía Electrónica , Fosforilación , Unión Proteica , Proteínas Serina-Treonina QuinasasRESUMEN
Nonsense-mediated decay (NMD) is a surveillance mechanism that degrades aberrant mRNAs. A complex comprising SMG1, UPF1, and the translation termination factors eRF1 and eRF3 (SURF) is assembled in the vicinity of a premature termination codon. Subsequently, an interaction with UPF2, UPF3b, and the exon junction complex induces the formation of the decay-inducing complex (DECID) and triggers NMD. We previously identified the RNA helicase DHX34 as an NMD factor in C. elegans and in vertebrates. Here, we investigate the mechanism by which DHX34 activates NMD in human cells. We show that DHX34 is recruited to the SURF complex via its preferential interaction with hypophosphorylated UPF1. A series of molecular transitions induced by DHX34 include enhanced recruitment of UPF2, increased UPF1 phosphorylation, and dissociation of eRF3 from UPF1. Thus, DHX34 promotes mRNP remodeling and triggers the conversion from the SURF complex to the DECID complex resulting in NMD activation.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , Factores de Terminación de Péptidos/metabolismo , ARN Helicasas/metabolismo , Células HEK293 , Humanos , Factores de Terminación de Péptidos/química , Fosforilación , Unión Proteica , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Ribonucleoproteínas/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The physical ends of chromosomes, known as telomeres, protect chromosome ends from nucleolytic degradation and DNA repair activities. Conventional DNA replication enzymes lack the ability to fully replicate telomere ends. In addition, nucleolytic activities contribute to telomere erosion. Short telomeres trigger DNA damage checkpoints, which mediate cellular senescence. Telomere length homeostasis requires telomerase, a cellular reverse transcriptase, which uses an internal RNA moiety as a template for the synthesis of telomere repeats. Telomerase elongates the 3' ends of chromosomes, whereas the complementary strand is filled in by conventional DNA polymerases. In humans, telomerase is ubiquitously expressed only during the first weeks of embryogenesis, and is subsequently downregulated in most cell types. Correct telomere length setting is crucial for long-term survival. The telomere length reserve must be sufficient to avoid premature cellular senescence and the acceleration of age-related disease. On the other side, telomere shortening suppresses tumor formation through limiting the replicative potential of cells. In recent years, novel insight into the regulation of telomerase at chromosome ends has increased our understanding on how telomere length homeostasis in telomerase-positive cells is achieved. Factors that recruit telomerase to telomeres in a cell cycle-dependent manner have been identified in Saccharomyces cerevisiae. In humans, telomerase assembles with telomeres during S phase of the cell cycle. Presumably through mediating formation of alternative telomere structures, telomere-binding proteins regulate telomerase activity in cis to favor preferential elongation of the shortest telomeres. Phosphoinositide 3-kinase related kinases are also required for telomerase activation at chromosome ends, at least in budding and fission yeast. In vivo analysis of telomere elongation kinetics shows that telomerase does not act on every telomere in each cell cycle but that it exhibits an increasing preference for telomeres as their lengths decline. This suggests a model in which telomeres switch between extendible and nonextendible states in a length-dependent manner. In this review we expand this model to incorporate the finding that telomerase levels also limit telomere length and we propose a second switch between a non-telomerase-associated "extendible" and a telomerase-associated "extending" state.