RESUMEN
3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.
Asunto(s)
Regiones no Traducidas 3'/genética , Evolución Biológica , Enfermedad/genética , Estudio de Asociación del Genoma Completo , Algoritmos , Alelos , Regulación de la Expresión Génica , Genes Reporteros , Variación Genética , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Polirribosomas/metabolismo , Sitios de Carácter Cuantitativo/genética , ARN/genéticaRESUMEN
Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
Asunto(s)
ADP-Ribosilación , Neoplasias Ováricas/metabolismo , Biosíntesis de Proteínas , Proteostasis , Ribosomas/metabolismo , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Estrés del Retículo Endoplásmico , Trompas Uterinas/metabolismo , Femenino , Humanos , Ratones Endogámicos NOD , Ratones SCID , NAD/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa , Conformación de Ácido Nucleico , Neoplasias Ováricas/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.
Asunto(s)
Ribosomas/metabolismo , Estrés Fisiológico , Transportadoras de Casetes de Unión a ATP/metabolismo , Anisomicina/farmacología , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de la radiación , Activación Enzimática , Humanos , Quinasas Quinasa Quinasa PAM/deficiencia , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Fosforilación , Polirribosomas/metabolismo , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Rayos Ultravioleta , eIF-2 Quinasa/metabolismoRESUMEN
Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Animales , Ribosomas/genética , Ribosomas/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , MamíferosRESUMEN
The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.
Asunto(s)
Citoplasma , Polirribosomas , Ribonucleoproteínas , Polirribosomas/metabolismo , Citoplasma/metabolismo , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Simulación de Dinámica Molecular , ARN Mensajero/metabolismo , ARN Mensajero/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Condensados Biomoleculares/metabolismo , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genéticaRESUMEN
Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.
Asunto(s)
Núcleo Celular , Cromatina , ARN Helicasas DEAD-box , ARN Mensajero , Animales , Humanos , Ratones , ARN Mensajero/metabolismo , ARN Mensajero/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Cromatina/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Citoplasma/genética , Estabilidad del ARN , Transporte Activo de Núcleo Celular , Polirribosomas/metabolismo , Polirribosomas/genética , Aprendizaje Automático , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Exosomas/metabolismo , Exosomas/genéticaRESUMEN
Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remain controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than â¼ 590 nt, and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Semivida , Degradación de ARNm Mediada por Codón sin Sentido , Polirribosomas/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/citologíaRESUMEN
tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.
Asunto(s)
Histona H2a Dioxigenasa, Homólogo 1 de AlkB/metabolismo , Regulación de la Expresión Génica , Biosíntesis de Proteínas/genética , ARN de Transferencia/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/genética , Glucosa/deficiencia , Células HeLa , Humanos , Metilación , Polirribosomas/metabolismoRESUMEN
Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites. Using this approach, we monitor transient changes of translation dynamics in responses to environmental stresses, capture distinct mobilities of individual polysomes in different subcellular compartments, and detect 3' UTR-dependent local translation and active transport of polysomes in dendrites of primary neurons.
Asunto(s)
Imagen Óptica/métodos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Dendritas/metabolismo , Humanos , Polirribosomas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/químicaRESUMEN
Among the three phases of mRNA translation-initiation, elongation, and termination-initiation has traditionally been considered to be rate limiting and thus the focus of regulation. Emerging evidence, however, demonstrates that control of ribosome translocation (polypeptide elongation) can also be regulatory and indeed exerts a profound influence on development, neurologic disease, and cell stress. The correspondence of mRNA codon usage and the relative abundance of their cognate tRNAs is equally important for mediating the rate of polypeptide elongation. Here, we discuss recent results showing that ribosome pausing is a widely used mechanism for controlling translation and, as a result, biological transitions in health and disease.
Asunto(s)
Regulación de la Expresión Génica , Extensión de la Cadena Peptídica de Translación , Polirribosomas/metabolismo , Animales , Codón , Enfermedad/genética , Humanos , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Regnase-1 and Roquin are RNA binding proteins essential for degradation of inflammation-related mRNAs and maintenance of immune homeostasis. However, their mechanistic relationship has yet to be clarified. Here, we show that, although Regnase-1 and Roquin regulate an overlapping set of mRNAs via a common stem-loop structure, they function in distinct subcellular locations: ribosome/endoplasmic reticulum and processing-body/stress granules, respectively. Moreover, Regnase-1 specifically cleaves and degrades translationally active mRNAs and requires the helicase activity of UPF1, similar to the decay mechanisms of nonsense mRNAs. In contrast, Roquin controls translationally inactive mRNAs, independent of UPF1. Defects in both Regnase-1 and Roquin lead to large increases in their target mRNAs, although Regnase-1 tends to control the early phase of inflammation when mRNAs are more actively translated. Our findings reveal that differential regulation of mRNAs by Regnase-1 and Roquin depends on their translation status and enables elaborate control of inflammation.
Asunto(s)
Inflamación/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Secuencia de Bases , Codón de Terminación , Células HeLa , Humanos , Inflamación/genética , Inflamación/inmunología , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Conformación de Ácido Nucleico , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/química , Proteínas Ribosómicas/metabolismo , Transactivadores/metabolismoRESUMEN
The unfolded protein response (UPR) is a stress response program that reprograms cellular translation and gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). One of the primary means by which the UPR alleviates this stress is by reducing protein flux into the ER via a general suppression of protein synthesis and ER-specific mRNA degradation. We report here an additional UPR-induced mechanism for the reduction of protein flux into the ER, where mRNAs that encode signal sequences are released from the ER to the cytosol. By removing mRNAs from the site of translocation, this mechanism may serve as a potent means to transiently reduce ER protein folding load and restore proteostasis. These findings identify the dynamic subcellular localization of mRNAs and translation as a selective and rapid regulatory feature of the cellular response to protein folding stress.
Asunto(s)
Retículo Endoplásmico/metabolismo , ARN Mensajero/metabolismo , Respuesta de Proteína Desplegada , Animales , Citosol/metabolismo , Ditiotreitol/metabolismo , Retículo Endoplásmico/química , Fibroblastos , Cinética , Ratones , Sistemas de Lectura Abierta , Polirribosomas/metabolismo , Biosíntesis de ProteínasRESUMEN
An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.
Asunto(s)
Ambystoma mexicanum , Evolución Biológica , Biosíntesis de Proteínas , Regeneración , Serina-Treonina Quinasas TOR , Animales , Humanos , Ratones , Ambystoma mexicanum/fisiología , Secuencia de Aminoácidos , Extremidades/fisiología , Regeneración/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Cicatrización de Heridas , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Especificidad de la Especie , Antioxidantes/metabolismo , Nutrientes/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismoRESUMEN
Translation inhibition is a major but poorly understood mode of action of microRNAs (miRNAs) in plants and animals. In particular, the subcellular location where this process takes place is unknown. Here, we show that the translation inhibition, but not the mRNA cleavage activity, of Arabidopsis miRNAs requires ALTERED MERISTEM PROGRAM1 (AMP1). AMP1 encodes an integral membrane protein associated with endoplasmic reticulum (ER) and ARGONAUTE1, the miRNA effector and a peripheral ER membrane protein. Large differences in polysome association of miRNA target RNAs are found between wild-type and the amp1 mutant for membrane-bound, but not total, polysomes. This, together with AMP1-independent recruitment of miRNA target transcripts to membrane fractions, shows that miRNAs inhibit the translation of target RNAs on the ER. This study demonstrates that translation inhibition is an important activity of plant miRNAs, reveals the subcellular location of this activity, and uncovers a previously unknown function of the ER.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Carboxipeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , MicroARNs/metabolismo , ARN de Planta/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Carboxipeptidasas/genética , Pleiotropía Genética , Mutación , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismoRESUMEN
Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells1. Here we use advances in cryo-electron tomography and sub-tomogram analysis2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes4. By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells.
Asunto(s)
Microscopía por Crioelectrón , Mycoplasma pneumoniae , Biosíntesis de Proteínas , Proteínas Ribosómicas , Ribosomas , Antibacterianos/farmacología , Mycoplasma pneumoniae/citología , Mycoplasma pneumoniae/efectos de los fármacos , Mycoplasma pneumoniae/metabolismo , Mycoplasma pneumoniae/ultraestructura , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Polirribosomas/efectos de los fármacos , Polirribosomas/metabolismo , Polirribosomas/ultraestructura , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/metabolismo , Complejos Multiproteicos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Sistema Libre de Células , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Conejos , Subunidades Ribosómicas/genética , Subunidades Ribosómicas/metabolismo , Ribosomas/genética , UbiquitinaciónRESUMEN
Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.
Asunto(s)
Flavivirus/crecimiento & desarrollo , Flavivirus/metabolismo , Interacciones Huésped-Patógeno , Hidroxilación , Procolágeno-Prolina Dioxigenasa/metabolismo , Prolina/metabolismo , Biosíntesis de Proteínas , Línea Celular , Colágeno/química , Colágeno/metabolismo , Virus del Dengue/genética , Virus del Dengue/crecimiento & desarrollo , Flavivirus/química , Regulación Viral de la Expresión Génica , Genómica , Factores Celulares Derivados del Huésped/antagonistas & inhibidores , Factores Celulares Derivados del Huésped/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Sitios Internos de Entrada al Ribosoma , Chaperonas Moleculares/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Poliovirus/genética , Poliovirus/crecimiento & desarrollo , Polirribosomas/química , Polirribosomas/metabolismo , Agregado de Proteínas , Pliegue de Proteína , Mapas de Interacción de Proteínas , Proteolisis , Proteómica , Virus Zika/genética , Virus Zika/crecimiento & desarrolloRESUMEN
In addition to their central functions in translation, ribosomes can adopt inactive structures that are fully assembled yet devoid of mRNA. We describe how the abundance of idle eukaryotic ribosomes is influenced by a broad range of biological conditions spanning viral infection, nutrient deprivation, and developmental cues. Vacant ribosomes may provide a means to exclude ribosomes from translation while also shielding them from degradation, and the variable identity of factors that occlude ribosomes may impart distinct functionality. We propose that regulated changes in the balance of idle and active ribosomes provides a means to fine-tune translation. We provide an overview of idle ribosomes, describe what is known regarding their function, and highlight questions that may clarify their biological roles.
Asunto(s)
Proteínas Ribosómicas , Ribosomas , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue-specific and essential for some tissue development, such as the liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by using a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in the transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays a more relevant role in small RNA metabolism and ribosome biogenesis.
Asunto(s)
Adenosina Desaminasa , Hepatocitos , Edición de ARN , Proteínas de Unión al ARN , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Hepatocitos/metabolismo , Polirribosomas/metabolismo , Polirribosomas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Biosíntesis de Proteínas , Transcriptoma , Técnicas de Inactivación de Genes , Línea CelularRESUMEN
FMRP loss of function causes Fragile X syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here, we use high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results suggest that loss of a translational brake on the synthesis of a subset of synaptic proteins contributes to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD and suggest multiple targets for clinical intervention.