RESUMEN
mRNA translation initiation plays a critical role in learning and memory. The eIF4F complex, composed of the cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffolding protein eIF4G, is a pivotal factor in the mRNA translation initiation process. eIF4G1, the major paralogue of the three eIF4G family members, is indispensable for development, but its function in learning and memory is unknown. To study the role of eIF4G1 in cognition, we used an eIF4G1 haploinsufficient (eIF4G1-1D) mouse model. The axonal arborization of eIF4G1-1D primary hippocampal neurons was significantly disrupted, and the mice displayed impairment in hippocampus-dependent learning and memory. Translatome analysis showed that the translation of mRNAs encoding proteins of the mitochondrial oxidative phosphorylation (OXPHOS) system was decreased in the eIF4G1-1D brain, and OXPHOS was decreased in eIF4G1-silenced cells. Thus, eIF4G1-mediated mRNA translation is crucial for optimal cognitive function, which is dependent on OXPHOS and neuronal morphogenesis.
Asunto(s)
Factor 4G Eucariótico de Iniciación , Fosforilación Oxidativa , Animales , Ratones , ARN Mensajero , Iniciación de la Cadena Peptídica Traduccional , Morfogénesis , ADN HelicasasRESUMEN
RATIONALE: mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression. METHODS: We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels. RESULTS: The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes. CONCLUSIONS: The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.
Asunto(s)
Proteínas Fluorescentes Verdes , Péptidos , ARN Mensajero , ARN Mensajero/genética , ARN Mensajero/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Péptidos/química , Péptidos/análisis , Péptidos/genética , Péptidos/metabolismo , Humanos , Espectrometría de Masas/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNA-protein complex (microRNP) that lacks the microRNP repressor, GW182. Their mechanism in these conditions of decreased mTOR signaling, and therefore reduced canonical (cap-and-poly(A)-tail-mediated) translation, remains undiscovered. Our data reveal that mTOR inhibition in human THP1 cells enables microRNA-mediated activation. Activation requires shortened/no poly(A)-tail targets; polyadenylated mRNAs are partially activated upon PAIP2 overexpression, which interferes with poly(A)-bound PABP, precluding PABP-enhanced microRNA-mediated inhibition and canonical translation. Consistently, inhibition of PARN deadenylase prevents activation. P97/DAP5, a homolog of canonical translation factor, eIF4G, which lacks PABP- and cap binding complex-interacting domains, is required for activation, and thereby for the oocyte immature state. P97 interacts with 3' UTR-binding FXR1a-associated microRNPs and with PARN, which binds mRNA 5' caps, forming a specialized complex to translate recruited mRNAs in these altered canonical translation conditions.
Asunto(s)
Senescencia Celular , MicroARNs/metabolismo , Oocitos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Regiones no Traducidas 3' , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sitios de Unión , Línea Celular , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , MicroARNs/genética , Proteómica/métodos , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Interferencia de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ribonucleoproteínas/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Transfección , Xenopus laevisRESUMEN
Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphoma-prone Eµ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e∆38 fs/+/Eµ-Myc or Eif4a1∆5 fs/+/Eµ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Factor 4F Eucariótico de Iniciación/genética , Animales , Femenino , Regulación de la Expresión Génica/genética , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Subunidades de Proteína/genética , Espermatogénesis/genéticaRESUMEN
Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.
Asunto(s)
Herpes Simple/patología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/genética , Neoplasias/virología , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Dominio Catalítico/efectos de los fármacos , Proteínas de Ciclo Celular , Células Cultivadas , Chlorocebus aethiops , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Herpes Simple/complicaciones , Herpes Simple/genética , Humanos , Proteínas Inmediatas-Precoces/deficiencia , Ratones , Neoplasias/complicaciones , Neoplasias/genética , Neoplasias/patología , Organismos Modificados Genéticamente , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/química , Ubiquitina-Proteína Ligasas/deficiencia , Células VeroRESUMEN
Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [Cnot7/8-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (Cnot6/6l-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8-dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l-dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8-dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Importantly, Cnot6/6l-dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.
Asunto(s)
Exorribonucleasas/metabolismo , ARN Mensajero/metabolismo , Receptores CCR4/metabolismo , Proteínas Represoras/metabolismo , Animales , Supervivencia Celular/genética , Exorribonucleasas/genética , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Masculino , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Poli A/genética , Poli A/metabolismo , Subunidades de Proteína , Estabilidad del ARN , ARN Mensajero/genética , Receptores CCR4/genética , Proteínas Represoras/genéticaRESUMEN
Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Proteínas de Unión a Caperuzas de ARN/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Ciclo Celular , Células HEK293 , Células HeLa , Homeostasis , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión a Caperuzas de ARN/metabolismo , Transfección , Ubiquitina/metabolismoRESUMEN
Translational control of gene expression plays a key role during the early phases of embryonic development. Here we describe a transcriptional regulator of mouse embryonic stem cells (mESCs), Yin-yang 2 (YY2), that is controlled by the translation inhibitors, Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays a critical role in regulating mESC functions through control of key pluripotency factors, including Octamer-binding protein 4 (Oct4) and Estrogen-related receptor-ß (Esrrb). Importantly, overexpression of YY2 directs the differentiation of mESCs into cardiovascular lineages. We show that the splicing regulator Polypyrimidine tract-binding protein 1 (PTBP1) promotes the retention of an intron in the 5'-UTR of Yy2 mRNA that confers sensitivity to 4E-BP-mediated translational suppression. Thus, we conclude that YY2 is a major regulator of mESC self-renewal and lineage commitment and document a multilayer regulatory mechanism that controls its expression.
Asunto(s)
Empalme Alternativo/fisiología , Diferenciación Celular , Autorrenovación de las Células/fisiología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/metabolismo , Animales , Blastocisto/metabolismo , Proteínas Portadoras/metabolismo , Linaje de la Célula , Autorrenovación de las Células/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Intrones , Ratones , Ratones Noqueados , Modelos Biológicos , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosfoproteínas , Proteína de Unión al Tracto de Polipirimidina/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Estrógenos/metabolismo , Factores de Transcripción/genética , Transcripción Genética/fisiología , Factor de Transcripción YY1/metabolismoRESUMEN
The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1.
Asunto(s)
Autoantígenos/metabolismo , Regulación hacia Abajo , Glicoproteínas de Membrana/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Ribonucleoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autoantígenos/genética , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Glicoproteínas de Membrana/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , ARN Largo no Codificante , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteína Reguladora Asociada a mTOR , Ribonucleoproteínas/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Antígeno SS-BRESUMEN
Translation control often operates via remodeling of messenger ribonucleoprotein particles. The poly(A) binding protein (PABP) simultaneously interacts with the 3' poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation. PABP also promotes miRNA-dependent deadenylation and translational repression of target mRNAs. We demonstrate that isoform 2 of the mouse heterogeneous nuclear protein Q (hnRNP-Q2/SYNCRIP) binds poly(A) by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to poly(A) in vitro. Depleting hnRNP-Q2 from translation extracts stimulates cap-dependent and IRES-mediated translation that is dependent on the PABP/poly(A) complex. Adding recombinant hnRNP-Q2 to the extracts inhibited translation in a poly(A) tail-dependent manner. The displacement of PABP from the poly(A) tail by hnRNP-Q2 impaired the association of eIF4E with the 5' m(7)G cap structure of mRNA, resulting in the inhibition of 48S and 80S ribosome initiation complex formation. In mouse fibroblasts, silencing of hnRNP-Q2 stimulated translation. In addition, hnRNP-Q2 impeded let-7a miRNA-mediated deadenylation and repression of target mRNAs, which require PABP. Thus, by competing with PABP, hnRNP-Q2 plays important roles in the regulation of global translation and miRNA-mediated repression of specific mRNAs.
Asunto(s)
Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , MicroARNs/metabolismo , Animales , Sitios de Unión , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Ratones , MicroARNs/genéticaRESUMEN
Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.
Asunto(s)
Cromosomas Humanos Par 3/genética , Factor 4G Eucariótico de Iniciación/genética , Enfermedad de Parkinson/genética , Biosíntesis de Proteínas/genética , Secuencia de Bases , Clonación Molecular , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Citometría de Flujo , Ligamiento Genético , Genotipo , Humanos , Inmunoprecipitación , Mitocondrias/fisiología , Datos de Secuencia Molecular , Mutación Missense/genética , LinajeRESUMEN
Persistent protein synthesis inhibition (PSI) is a robust predictor of eventual neuronal death following cerebral ischemia. We thus tested the hypothesis that persistent PSI inhibition and neuronal death are causally linked. Neuronal viability strongly correlated with both protein synthesis and levels of eukaryotic (translation) initiation factor 4G1 (eIF4G1). We determined that in vitro ischemia activated calpain, which degraded eIF4G1. Overexpression of the calpain inhibitor calpastatin or eIF4G1 resulted in increased protein synthesis and increased neuronal viability compared with controls. The neuroprotective effect of eIF4G1 overexpression was due to restoration of cap-dependent protein synthesis, as well as protein synthesis-independent mechanisms, as inhibition of protein synthesis with cycloheximide did not completely prevent the protective effect of eIF4G1 overexpression. In contrast, shRNA-mediated silencing of eIF4G1 exacerbated ischemia-induced neuronal injury, suggesting eIF4G1 is necessary for maintenance of neuronal viability. Finally, calpain inhibition following global ischemia in vivo blocked decreases in eIF4G1, facilitated protein synthesis, and increased neuronal viability in ischemia-vulnerable hippocampal CA1 neurons. Collectively, these data demonstrate that calpain-mediated degradation of a translation initiation factor, eIF4G1, is a cause of both persistent PSI and neuronal death.
Asunto(s)
Calpaína/metabolismo , Muerte Celular , Factor 4G Eucariótico de Iniciación/metabolismo , Isquemia/enzimología , Neuronas/metabolismo , Inhibidores de la Síntesis de la Proteína , Animales , Calpaína/antagonistas & inhibidores , Activación Enzimática , Factor 4G Eucariótico de Iniciación/genética , Silenciador del Gen , Neuronas/citología , Neuronas/enzimología , Biosíntesis de Proteínas , Caperuzas de ARN , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Mammalian circadian rhythms are synchronized to the external time by daily resetting of the suprachiasmatic nucleus (SCN) in response to light. As the master circadian pacemaker, the SCN coordinates the timing of diverse cellular oscillators in multiple tissues. Aberrant regulation of clock timing is linked to numerous human conditions, including cancer, cardiovascular disease, obesity, various neurological disorders and the hereditary disorder familial advanced sleep phase syndrome. Additionally, mechanisms that underlie clock resetting factor into the sleep and physiological disturbances experienced by night-shift workers and travelers with jet lag. The Ca(2+)/cAMP response element-binding protein-regulated microRNA, miR-132, is induced by light within the SCN and attenuates its capacity to reset, or entrain, the clock. However, the specific targets that are regulated by miR-132 and underlie its effects on clock entrainment remained elusive until now. Here, we show that genes involved in chromatin remodeling (Mecp2, Ep300, Jarid1a) and translational control (Btg2, Paip2a) are direct targets of miR-132 in the mouse SCN. Coordinated regulation of these targets underlies miR-132-dependent modulation of Period gene expression and clock entrainment: the mPer1 and mPer2 promoters are bound to and transcriptionally activated by MeCP2, whereas PAIP2A and BTG2 suppress the translation of the PERIOD proteins by enhancing mRNA decay. We propose that miR-132 is selectively enriched for chromatin- and translation-associated target genes and is an orchestrator of chromatin remodeling and protein translation within the SCN clock, thereby fine-tuning clock entrainment. These findings will further our understanding of mechanisms governing clock entrainment and its involvement in human diseases.
Asunto(s)
Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Ritmo Circadiano/genética , MicroARNs/genética , MicroARNs/metabolismo , Animales , Biología Computacional , Proteínas de Unión al ADN , Proteína p300 Asociada a E1A/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Histona Demetilasas con Dominio de Jumonji , Luz , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Estabilidad del ARN , Proteína 2 de Unión a Retinoblastoma/metabolismo , Transducción de Señal , Núcleo Supraquiasmático/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factor 4F Eucariótico de Iniciación/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Biosíntesis de Proteínas , Animales , Extractos Celulares , Línea Celular , Ratones , Modelos Biológicos , Conejos , Proteína 1 de Unión a la Caja YRESUMEN
Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD(1408)S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.
Asunto(s)
Factor 4G Eucariótico de Iniciación/metabolismo , Granzimas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Linfocitos T Citotóxicos/metabolismo , Virus Vaccinia/metabolismo , Replicación Viral , Apoptosis/fisiología , Caspasa 3/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Células Jurkat , Proteolisis , Proteómica , Virus Vaccinia/fisiología , Replicación Viral/fisiologíaRESUMEN
Translation initiation in eukaryotes is regulated at several steps, one of which involves the availability of the cap binding protein to participate in cap-dependent protein synthesis. Binding of eIF4E to translational repressors (eIF4E-binding proteins [4E-BPs]) suppresses translation and is used by cells to link extra- and intracellular cues to protein synthetic rates. The best studied of these interactions involves repression of translation by 4E-BP1 upon inhibition of the PI3K/mTOR signaling pathway. Herein, we characterize a novel 4E-BP, C8ORF88, whose expression is predominantly restricted to early spermatids. C8ORF88:eIF4E interaction is dependent on the canonical eIF4E binding motif (4E-BM) present in other 4E-BPs. Whereas 4E-BP1:eIF4E interaction is dependent on the phosphorylation of 4E-BP1, these sites are not conserved in C8ORF88 indicating a different mode of regulation.
Asunto(s)
Proteínas Portadoras , Factor 4E Eucariótico de Iniciación , Proteínas Portadoras/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , FosforilaciónRESUMEN
During spermiogenesis, expression of the specific proteins needed for proper differentiation of male germ cells is under translational control. We have shown that PAIP2A is a major translational regulator involved in the maturation of male germ cells and male fertility. To identify the proteins controlled by PAIP2A during spermiogenesis, we characterized the proteomic profiles of elongated spermatids from wild-type (WT) mice and mice that were Paip2a/Paip2b double-null mutants (DKO). Elongated spermatid populations were obtained and proteins were extracted and separated on gradient polyacrylamide gels. The gels were digested with trypsin and peptides were identified by mass spectrometry. We identified 632 proteins with at least two unique peptides and a confidence level of 95%. Only 209 proteins were consistently detected in WT or DKO replicates with more than five spectra. Twenty-nine proteins were differentially expressed with at least a 1.5-fold change; 10 and 19 proteins were down- and up-regulated, respectively, in DKO compared to WT mice. We confirmed the significantly different expression levels of three proteins, EIF4G1, AKAP4, and HK1, by Western blot analysis. We have characterized novel proteins that have their expression controlled by PAIP2A; of these, 50% are involved in flagellar structure and sperm motility. Although several proteins affected by abrogation of Paip2a have established roles in reproduction, the roles of many others remain to be determined.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/fisiología , Proteínas Portadoras/fisiología , Factor 4G Eucariótico de Iniciación/fisiología , Hexoquinasa/fisiología , Espermatogénesis/fisiología , Proteínas de Anclaje a la Quinasa A/genética , Animales , Proteínas Portadoras/genética , Factor 4G Eucariótico de Iniciación/genética , Fertilidad/genética , Fertilidad/fisiología , Flagelos/fisiología , Perfilación de la Expresión Génica , Hexoquinasa/genética , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Proteómica , Proteínas de Unión al ARN , Proteínas Represoras , Motilidad Espermática/fisiología , Espermatogénesis/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
The CCR4-NOT deadenylase complex is a major post-transcriptional regulator of eukaryotic gene expression. CNOT7 and CNOT8 are both vertebrate homologs of the yeast CCR4-NOT catalytic subunit Caf1. They are highly similar and are sometimes considered redundant, but Cnot7 and Cnot8 knockout mice exhibit different phenotypes, implying distinct physiological functions. In this study, we reveal a non-reciprocal effect of CNOT7 on CNOT8, in which CNOT8 protein is increased in the depletion of CNOT7 without corresponding changes in mRNA levels whereas CNOT7 is not affected by the loss of CNOT8. Cnot8 mRNA may be bound by the CCR4-NOT complex, suggesting that CCR4-NOT might directly regulate CNOT8 expression. Cnot8 mRNA is relatively unstable, but Cnot7 knockdown did not stabilize Cnot8 mRNA, nor did it increase translation. CNOT8 protein was also less stable than CNOT7. CNOT7 showed greater affinity than CNOT8 for the CCR4-NOT scaffold protein CNOT1 and was able to block CNOT8 from binding to CNOT1. Depletion of CNOT7 increased CNOT8 incorporation into the CCR4-NOT complex and stabilized CNOT8. These data suggest that CNOT7 is the dominant paralog in CCR4-NOT and that CNOT7 and CNOT8 protein stability is regulated in distinct ways.
Asunto(s)
Exorribonucleasas , Proteínas Represoras , Factores de Transcripción , Animales , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Ribonucleasas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Control of mRNA stability and degradation is essential for appropriate gene expression, and its dysregulation causes various disorders, including cancer, neurodegenerative diseases, diabetes, and obesity. The 5'-3' exoribonuclease XRN1 executes the last step of RNA decay, but its physiological impact is not well understood. To address this, forebrain-specific Xrn1 conditional knockout mice (Xrn1-cKO) were generated, as Xrn1 null mice were embryonic lethal. Xrn1-cKO mice exhibited obesity with leptin resistance, hyperglycemia, hyperphagia, and decreased energy expenditure. Obesity resulted from dysregulated communication between the central nervous system and peripheral tissues. Moreover, expression of mRNAs encoding proteins that regulate appetite and energy expenditure was dysregulated in the hypothalamus of Xrn1-cKO mice. Therefore, we propose that XRN1 function in the hypothalamus is critical for maintenance of metabolic homeostasis.
RESUMEN
Pancreatic ß-cells are responsible for production and secretion of insulin in response to increasing blood glucose levels. Defects in ß-cell function lead to hyperglycemia and diabetes mellitus. Here, we show that CNOT3, a CCR4-NOT deadenylase complex subunit, is dysregulated in islets in diabetic db/db mice, and that it is essential for murine ß cell maturation and identity. Mice with ß cell-specific Cnot3 deletion (Cnot3ßKO) exhibit impaired glucose tolerance, decreased ß cell mass, and they gradually develop diabetes. Cnot3ßKO islets display decreased expression of key regulators of ß cell maturation and function. Moreover, they show an increase of progenitor cell markers, ß cell-disallowed genes, and genes relevant to altered ß cell function. Cnot3ßKO islets exhibit altered deadenylation and increased mRNA stability, partly accounting for the increased expression of those genes. Together, these data reveal that CNOT3-mediated mRNA deadenylation and decay constitute previously unsuspected post-transcriptional mechanisms essential for ß cell identity.