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[This corrects the article DOI: 10.1371/journal.pbio.3002355.].
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Gtf2i encodes the general transcription factor II-I (TFII-I), with peak expression during pre-natal and early post-natal brain development stages. Because these stages are critical for proper brain development, we studied at the single-cell level the consequences of Gtf2i's deletion from excitatory neurons, specifically on mitochondria. Here we show that Gtf2i's deletion resulted in abnormal morphology, disrupted mRNA related to mitochondrial fission and fusion, and altered autophagy/mitophagy protein expression. These changes align with elevated reactive oxygen species levels, illuminating Gtf2i's importance in neurons mitochondrial function. Similar mitochondrial issues were demonstrated by Gtf2i heterozygous model, mirroring the human condition in Williams syndrome (WS), and by hemizygous neuronal Gtf2i deletion model, indicating Gtf2i's dosage-sensitive role in mitochondrial regulation. Clinically relevant, we observed altered transcript levels related to mitochondria, hypoxia, and autophagy in frontal cortex tissue from WS individuals. Our study reveals mitochondrial and autophagy-related deficits shedding light on WS and other Gtf2i-related disorders.
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Factores de Transcripción TFIII , Síndrome de Williams , Humanos , Autofagia/genética , Heterocigoto , Neuronas/metabolismo , Factores de Transcripción TFIII/genética , Factores de Transcripción TFIII/metabolismo , Síndrome de Williams/genética , Síndrome de Williams/metabolismoRESUMEN
The introduction of premature termination codons (PTCs), as a result of splicing defects, insertions, deletions, or point mutations (also termed nonsense mutations), lead to numerous genetic diseases, ranging from rare neuro-metabolic disorders to relatively common inheritable cancer syndromes and muscular dystrophies. Over the years, a large number of studies have demonstrated that certain antibiotics and other synthetic molecules can act as PTC suppressors by inducing readthrough of nonsense mutations, thereby restoring the expression of full-length proteins. Unfortunately, most PTC readthrough-inducing agents are toxic, have limited effects, and cannot be used for therapeutic purposes. Thus, further efforts are required to improve the clinical outcome of nonsense mutation suppressors. Here, by focusing on enhancing readthrough of pathogenic nonsense mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, we show that disturbing the protein translation initiation complex, as well as targeting other stages of the protein translation machinery, enhances both antibiotic and non-antibiotic-mediated readthrough of nonsense mutations. These findings strongly increase our understanding of the mechanisms involved in nonsense mutation readthrough and facilitate the development of novel therapeutic targets for nonsense suppression to restore protein expression from a large variety of disease-causing mutated transcripts.
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Codón sin Sentido , Neoplasias , Humanos , Codón sin Sentido/genética , Biosíntesis de Proteínas/genética , Antibacterianos/farmacologíaRESUMEN
Regulation of mRNA translation in astrocytes gains a growing interest. However, until now, successful ribosome profiling of primary astrocytes has not been reported. Here, we optimized the standard 'polysome profiling' method and generated an effective protocol for polyribosome extraction, which enabled genome-wide assessment of mRNA translation dynamics along the process of astrocyte activation. Transcriptome (RNAseq) and translatome (Riboseq) data generated at 0, 24 and 48 h after cytokines treatment, revealed dynamic genome-wide changes in the expression level of â¼12 000 genes. The data clarify whether a change in protein synthesis rate results from a change in mRNA level or translation efficiency per se. It exhibit different expression strategies, based on changes in mRNA abundance and/or translation efficiency, which are specifically assigned to gene subsets depending on their function. Moreover, the study raises an important take-home message related to the possible presence of 'difficult to extract' polyribosome sub-groups, in all cell types, thus illuminating the impact of ribosomes extraction methodology on experiments addressing translation regulation.
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Astrocitos , Biosíntesis de Proteínas , Astrocitos/metabolismo , Polirribosomas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Perfilación de la Expresión Génica/métodosRESUMEN
Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1-dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.
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Enfermedades Desmielinizantes , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Sustancia Blanca , Animales , Apoptosis/genética , Enfermedades Desmielinizantes/patología , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Oligodendroglía/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sustancia Blanca/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Translation initiation factor 2B (eIF2B) is a master regulator of global protein synthesis in all cell types. The mild genetic Eif2b5(R132H) mutation causes a slight reduction in eIF2B enzymatic activity which leads to abnormal composition of mitochondrial electron transfer chain complexes and impaired oxidative phosphorylation. Previous work using primary fibroblasts isolated from Eif2b5(R132H/R132H) mice revealed that owing to increased mitochondrial biogenesis they exhibit normal cellular ATP level. In contrast to fibroblasts, here we show that primary astrocytes isolated from Eif2b5(R132H/R132H) mice are unable to compensate for their metabolic impairment and exhibit chronic state of low ATP level regardless of extensive adaptation efforts. Mutant astrocytes are hypersensitive to oxidative stress and to further energy stress. Moreover, they show migration deficit upon exposure to glucose starvation. The mutation in Eif2b5 prompts reactive oxygen species (ROS)-mediated inferior ability to stimulate the AMP-activated protein kinase (AMPK) axis, due to a requirement to increase the mammalian target of rapamycin complex-1 (mTORC1) signalling in order to enable oxidative glycolysis and generation of specific subclass of ROS-regulating proteins, similar to cancer cells. The data disclose the robust impact of eIF2B on metabolic and redox homeostasis programs in astrocytes and point at their hyper-sensitivity to mutated eIF2B. Thereby, it illuminates the central involvement of astrocytes in Vanishing White Matter Disease (VWMD), a genetic neurodegenerative leukodystrophy caused by homozygous hypomorphic mutations in genes encoding any of the 5 subunits of eIF2B.
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Adenosina Trifosfato/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Metabolismo Energético , Factor 2B Eucariótico de Iniciación/metabolismo , Leucoencefalopatías/metabolismo , Mitocondrias/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis , Astrocitos/patología , Encéfalo/patología , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Factor 2B Eucariótico de Iniciación/genética , Leucoencefalopatías/genética , Leucoencefalopatías/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Mitocondrias/genética , Mitocondrias/patología , Mutación , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Data normalization is a critical step in RNA sequencing (RNA-seq) analysis, aiming to remove systematic effects from the data to ensure that technical biases have minimal impact on the results. Analyzing numerous RNA-seq datasets, we detected a prevalent sample-specific length effect that leads to a strong association between gene length and fold-change estimates between samples. This stochastic sample-specific effect is not corrected by common normalization methods, including reads per kilobase of transcript length per million reads (RPKM), Trimmed Mean of M values (TMM), relative log expression (RLE), and quantile and upper-quartile normalization. Importantly, we demonstrate that this bias causes recurrent false positive calls by gene-set enrichment analysis (GSEA) methods, thereby leading to frequent functional misinterpretation of the data. Gene sets characterized by markedly short genes (e.g., ribosomal protein genes) or long genes (e.g., extracellular matrix genes) are particularly prone to such false calls. This sample-specific length bias is effectively removed by the conditional quantile normalization (cqn) and EDASeq methods, which allow the integration of gene length as a sample-specific covariate. Consequently, using these normalization methods led to substantial reduction in GSEA false results while retaining true ones. In addition, we found that application of gene-set tests that take into account gene-gene correlations attenuates false positive rates caused by the length bias, but statistical power is reduced as well. Our results advocate the inspection and correction of sample-specific length biases as default steps in RNA-seq analysis pipelines and reiterate the need to account for intergene correlations when performing gene-set enrichment tests to lessen false interpretation of transcriptomic data.
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ARN/química , Análisis de Secuencia de ARN/normas , Animales , Sesgo , Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Humanos , Ratones , TranscriptomaRESUMEN
Vanishing white matter (VWM) disease (OMIM#306896) is an autosomal recessive neurodegenerative leukodystrophy caused by hypomorphic mutations in any of the five genes encoding the subunits of eukaryotic translation initiation factor 2B (eIF2B). The disease is manifested by loss of cerebral white matter and progressive deterioration upon exposure to environmental and physiological stressors. "Foamy" oligodendrocytes (OLG), increased numbers of oligodendrocytes precursor cells (OPC), and immature defective astrocytes are major neuropathological denominators. Our recent work using Eif2b5R132H/R132H mice uncovered a fundamental link between eIF2B and mitochondrial function. A decrease in oxidative phosphorylation capacity was observed in mutant astrocytes and fibroblasts. While an adaptive increase in mitochondria abundance corrects the phenotype of mutant fibroblasts, it is not sufficient to compensate for the high-energy demand of astrocytes, explaining their involvement in the disease. To date, astrocytes are marked as central for the disease while eIF2B-mutant OLG are currently assumed to lack a cellular phenotype on their own. Here we show a reduced capacity of eIF2B-mutant OPC isolated from Eif2b5R132H/R132H mice to conduct oxidative respiration despite the adaptive increase in their mitochondrial abundance. We also show their impaired ability to efficiently complete critical differentiation steps towards mature OLG. The concept that defective differentiation of eIF2B-mutant OPC could be a consequence of mitochondrial malfunction is in agreement with numerous studies indicating high dependency of differentiating OLG on accurate mitochondrial performance and ATP availability.
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Leucoencefalopatías/genética , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , Oligodendroglía/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Leucoencefalopatías/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Proteínas del Tejido Nervioso/fisiología , Fosforilación Oxidativa , Consumo de Oxígeno , Mutación Puntual , Proteoma , TranscriptomaRESUMEN
Vanishing white matter (VWM) disease is an autosomal genetic leukodystrophy caused by mutations in subunits of eukaryotic translation initiation factor 2B (eIF2B). The clinical symptoms exhibit progressive loss of white matter in both hemispheres of the brain, accompanied by motor functions deterioration, neurological deficits, and early death. To date there is no treatment for VWM disease. The aim of this work was to expedite rational development of a therapeutic opportunity. Our approach was to design a computer-aided strategy for an efficient and reliable screening of drug-like molecules; and to use primary cultures of fibroblasts isolated from the Eif2b5R132H/R132H VWM mouse model for screening. The abnormal mitochondria content phenotype of the mutant cells was chosen as a read-out for a simple cell-based fluorescent assay to assess the effect of the tested compounds. We obtained a hit rate of 0.04% (20 hits out of 50,000 compounds from the selected library). All primary hits decreased mitochondria content and brought it closer to WT levels. Structural similarities between our primary hits and other compounds with known targets allowed the identification of three putative cellular pathways/targets: 11ß-hydroxysteroid dehydrogenase type 1, Sonic hedgehog (Shh), and Sigma-1-Receptor (S1R). In addition to initial experimental indication of Shh pathway impairment in VWM mouse brains, the current study provides evidence that S1R is a relevant target for pharmaceutical intervention for potential treatment of the disease. Specifically, we found lower expression level of S1R protein in fibroblasts, astrocytes, and whole brains isolated from Eif2b5R132H/R132H compared to WT mice, and confirmed that one of the hits is a direct binder of S1R, acting as agonist. Furthermore, we provide evidence that treatment of mutant mouse fibroblasts and astrocytes with various S1R agonists corrects the functional impairments of their mitochondria and prevents their need to increase their mitochondria content for compensation purposes. Moreover, S1R activation enhances the survival rate of mutant cells under ER stress conditions, bringing it to WT levels. This study marks S1R as a target for drug development toward treatment of VWM disease. Moreover, it further establishes the important connection between white matter well-being and S1R-mediated proper mitochondria/ER function.
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The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.
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Estrés del Retículo Endoplásmico , Factor 3 de Iniciación Eucariótica/metabolismo , Fibroblastos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Transcripción Genética , eIF-2 Quinasa/metabolismo , Animales , Reprogramación Celular , Factor 3 de Iniciación Eucariótica/genética , Fibroblastos/patología , Células HEK293 , Humanos , Ratones , Sistemas de Lectura Abierta , Fenotipo , Proteostasis , Interferencia de ARN , ARN Mensajero/genética , Transducción de Señal , Factores de Tiempo , Transfección , eIF-2 Quinasa/genéticaRESUMEN
Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase.
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Mitosis/genética , Polirribosomas/fisiología , Biosíntesis de Proteínas/fisiología , Proteómica/métodos , Factores de Empalme de ARN/fisiología , Cromatografía Liquida , Fase G1 , Técnicas de Silenciamiento del Gen , Ontología de Genes , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo C/antagonistas & inhibidores , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo C/fisiología , Humanos , Interfase , Interferencia de ARN , Factores de Empalme de ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/fisiología , Proteínas Ribosómicas/genética , Espectrometría de Masas en TándemRESUMEN
Eukaryotic translation initiation factor 2B (eIF2B) is a master regulator of protein synthesis under normal and stress conditions. Mutations in any of the five genes encoding its subunits lead to vanishing white matter (VWM) disease, a recessive genetic deadly illness caused by progressive loss of white matter in the brain. In this study we used fibroblasts, which are not involved in the disease, to demonstrate the involvement of eIF2B in mitochondrial function and abundance. Mass spectrometry of total proteome of mouse embryonic fibroblasts (MEFs) isolated from Eif2b5R132H/R132H mice revealed unbalanced stoichiometry of proteins involved in oxidative phosphorylation and of mitochondrial translation machinery components, among others. Mutant MEFs exhibit 55% decrease in oxygen consumption rate per mtDNA content and 47% increase in mitochondrial abundance (p < 0.005), reflecting adaptation to energy requirements. A more robust eIF2B-associated oxidative respiration deficiency was found in mutant primary astrocytes, which exhibit > 3-fold lower ATP-linked respiration per cell despite a 2-fold increase in mtDNA content (p < 0.03). The 2-fold increase in basal and stimulated glycolysis in mutant astrocytes (p ≤ 0.03), but not in MEFs, demonstrates their higher energetic needs and further explicates their involvement in the disease. The data demonstrate the critical role of eIF2B in tight coordination of expression from nuclear and mitochondrial genomes and illuminates the importance of mitochondrial function in VWM pathology. Further dissection of the signaling network associated with eIF2B function will help generating therapeutic strategies for VWM disease and possibly other neurodegenerative disorders.
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Astrocitos/ultraestructura , Factor 2B Eucariótico de Iniciación/genética , Mitocondrias/genética , Mutación/genética , Fosforilación Oxidativa , Consumo de Oxígeno/genética , Animales , Animales Recién Nacidos , Antimicina A/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Tamaño de la Célula , Células Cultivadas , Cloranfenicol/farmacología , Factor 2B Eucariótico de Iniciación/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/ultraestructura , Antígenos de Histocompatibilidad/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/fisiología , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Ionóforos de Protónes/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression.
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Biosíntesis de Proteínas , Proteolisis , ARN Mensajero/biosíntesis , Transcripción Genética , División Celular/genética , Regulación de la Expresión Génica , Células HeLa , Humanos , Biosíntesis de Proteínas/genética , Proteoma/genética , ARN Mensajero/genética , Transcriptoma/genéticaRESUMEN
Vanishing white matter (VWM) is a recessive neurodegenerative disease caused by mutations in translation initiation factor eIF2B and leading to progressive brain myelin deterioration, secondary axonal damage, and death in early adolescence. Eif2b5(R132H/R132H) mice exhibit delayed developmental myelination, mild early neurodegeneration and a robust remyelination defect in response to cuprizone-induced demyelination. In the current study we used Eif2b5(R132H/R132H) mice for mass-spectrometry analyses, to follow the changes in brain protein abundance in normal- versus cuprizone-diet fed mice during the remyelination recovery phase. Analysis of proteome profiles suggested that dysregulation of mitochondrial functions, altered proteasomal activity and impaired balance between protein synthesis and degradation play a role in VWM pathology. Consistent with these findings, we detected elevated levels of reactive oxygen species in mutant-derived primary fibroblasts and reduced 20S proteasome activity in mutant brain homogenates. These observations highlight the importance of tight translational control to precise coordination of processes involved in myelin formation and regeneration and point at cellular functions that may contribute to VWM pathology. Eif2b5(R132H/R132H) mouse model for vanishing white matter (VWM) disease was used for mass spectrometry of brain proteins at two time points under normal conditions and along recovery from cuprizone-induced demyelination. Comparisons of proteome profiles revealed the importance of mitochondrial function and tight coordination between protein synthesis and degradation to myelination formation and regeneration, pointing at cellular functions that contribute to VWM pathology.
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Leucoencefalopatías/metabolismo , Vaina de Mielina/metabolismo , Regeneración Nerviosa/fisiología , Proteómica/métodos , Animales , Modelos Animales de Enfermedad , Factor 2B Eucariótico de Iniciación/genética , Leucoencefalopatías/genética , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones MutantesRESUMEN
Regulation of mRNA translation has a pivotal role in modulating protein levels, and the genome-wide identification of proteins synthesized at a given time is indispensable to our understanding of gene expression. This protocol describes the mass-spectrometric analysis of newly synthesized proteins from cultured cells or whole tissues by using a biotinylated derivative of puromycin, which becomes incorporated into nascent polypeptide chains by ribosome catalysis. In this method, termed puromycin-associated nascent chain proteomics (PUNCH-P), intact ribosome-nascent chain complexes are first recovered from cells by ultracentrifugation, followed by biotin-puromycin labeling of newly synthesized proteins, streptavidin affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unlike methods that require in vivo labeling, the sensitivity and coverage of PUNCH-P depend only on the amount of starting material and not on the duration of labeling, thus enabling the measurement of rapid fluctuations in protein synthesis. The protocol requires 3 d for sample preparation and analysis.
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Biosíntesis de Proteínas/genética , Proteómica/métodos , Puromicina/química , Espectrometría de Masas en Tándem/métodos , Biotina/química , Células Cultivadas , Cromatografía Liquida , Ribosomas/genética , UltracentrifugaciónRESUMEN
Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.
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Ciclo Celular/genética , Regulación del Desarrollo de la Expresión Génica , Proteómica/métodos , ARN Mensajero/metabolismo , Animales , Encéfalo/metabolismo , Células HeLa , Humanos , Espectrometría de Masas , Ratones , Proteínas/química , Proteómica/normas , Reproducibilidad de los Resultados , Ribosomas/metabolismoRESUMEN
The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA(Ile)UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Biosíntesis de Proteínas , Proteínas Virales/biosíntesis , Animales , Células CHO , Células Cultivadas , Codón , Cricetinae , Cricetulus , Virus de la Enfermedad Hemorrágica Epizoótica , Interferencia de ARN , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Análisis de la Célula Individual , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/genéticaRESUMEN
Regulation of mRNA translation is a major modulator of gene expression, allowing cells to fine tune protein levels during growth and differentiation and in response to physiological signals and environmental changes. Mass-spectrometry and RNA-sequencing methods now enable global profiling of the translatome, but these still involve significant analytical and economical limitations. We developed a novel system-wide proteomic approach for direct monitoring of translation, termed PUromycin-associated Nascent CHain Proteomics (PUNCH-P), which is based on the recovery of ribosome-nascent chain complexes from cells or tissues followed by incorporation of biotinylated puromycin into newly-synthesized proteins. Biotinylated proteins are then purified by streptavidin and analyzed by mass-spectrometry. Here we present an overview of PUNCH-P, describe other methodologies for global translatome profiling (pSILAC, BONCAT, TRAP/Ribo-tag, Ribo-seq) and provide conceptual comparisons between these methods. We also show how PUNCH-P data can be combined with mRNA measurements to determine relative translation efficiency for specific mRNAs.