RESUMO
Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/química , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica , Humanos , Camundongos Endogâmicos NOD , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Oncogenes , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Tamoxifeno/farmacologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the eIF4E dose requirement at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we found that a 50% reduction in eIF4E expression, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for the dose of eIF4E, specifically in translating a network of mRNAs enriched for a unique 5' UTR signature. In particular, we demonstrate that the dose of eIF4E is essential for translating mRNAs that regulate reactive oxygen species, fueling transformation and cancer cell survival in vivo. Our findings indicate eIF4E is maintained at levels in excess for normal development that are hijacked by cancer cells to drive a translational program supporting tumorigenesis.
Assuntos
Transformação Celular Neoplásica , Embrião de Mamíferos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Dosagem de Genes , Regiões 5' não Traduzidas , Animais , Carcinogênese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biossíntese de Proteínas , Espécies Reativas de Oxigênio/metabolismoRESUMO
Fasting is associated with a range of health benefits1-6. How fasting signals elicit changes in the proteome to establish metabolic programmes remains poorly understood. Here we show that hepatocytes selectively remodel the translatome while global translation is paradoxically downregulated during fasting7,8. We discover that phosphorylation of eukaryotic translation initiation factor 4E (P-eIF4E) is induced during fasting. We show that P-eIF4E is responsible for controlling the translation of genes involved in lipid catabolism and the production of ketone bodies. Inhibiting P-eIF4E impairs ketogenesis in response to fasting and a ketogenic diet. P-eIF4E regulates those messenger RNAs through a specific translation regulatory element within their 5' untranslated regions (5' UTRs). Our findings reveal a new signalling property of fatty acids, which are elevated during fasting. We found that fatty acids bind and induce AMP-activated protein kinase (AMPK) kinase activity that in turn enhances the phosphorylation of MAP kinase-interacting protein kinase (MNK), the kinase that phosphorylates eIF4E. The AMPK-MNK-eIF4E axis controls ketogenesis, revealing a new lipid-mediated kinase signalling pathway that links ketogenesis to translation control. Certain types of cancer use ketone bodies as an energy source9,10 that may rely on P-eIF4E. Our findings reveal that on a ketogenic diet, treatment with eFT508 (also known as tomivosertib; a P-eIF4E inhibitor) restrains pancreatic tumour growth. Thus, our findings unveil a new fatty acid-induced signalling pathway that activates selective translation, which underlies ketogenesis and provides a tailored diet intervention therapy for cancer.
RESUMO
In this issue of Molecular Cell, Liu et al. (2022) report that 5'-tRFCys, a stress-induced transfer RNA-derived RNA fragment (tRF) derived from the 5' halves of cysteine tRNAs, regulates post-transcriptional gene expression, enabling the survival and lung metastasis formation of breast cancers.
Assuntos
Neoplasias da Mama , Proteínas de Ligação a RNA , Neoplasias da Mama/genética , Feminino , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , NucleolinaRESUMO
The concept of specialized ribosomes has garnered equal amounts of interest and skepticism since it was first introduced. We ask researchers in the field to provide their perspective on the topic and weigh in on the evidence (or lack thereof) and what the future may bring.
Assuntos
Biossíntese de Proteínas , Ribossomos , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Activated CD8+ T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct T cell fate trajectories can be established during the first division.
Assuntos
Linfócitos T CD8-Positivos , Fator de Iniciação 4F em Eucariotos , Diferenciação Celular , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina/genéticaRESUMO
Cancer cells must integrate multiple biosynthetic demands to drive indefinite proliferation. How these key cellular processes, such as metabolism and protein synthesis, crosstalk to fuel cancer cell growth is unknown. Here, we uncover the mechanism by which the Myc oncogene coordinates the production of the two most abundant classes of cellular macromolecules, proteins, and nucleic acids in cancer cells. We find that a single rate-limiting enzyme, phosphoribosyl-pyrophosphate synthetase 2 (PRPS2), promotes increased nucleotide biosynthesis in Myc-transformed cells. Remarkably, Prps2 couples protein and nucleotide biosynthesis through a specialized cis-regulatory element within the Prps2 5' UTR, which is controlled by the oncogene and translation initiation factor eIF4E downstream Myc activation. We demonstrate with a Prps2 knockout mouse that the nexus between protein and nucleotide biosynthesis controlled by PRPS2 is crucial for Myc-driven tumorigenesis. Together, these studies identify a translationally anchored anabolic circuit critical for cancer cell survival and an unexpected vulnerability for "undruggable" oncogenes, such as Myc. PAPERFLICK:
Assuntos
Carcinogênese , Nucleotídeos/biossíntese , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribose-Fosfato Pirofosfoquinase/genética , Regiões 5' não Traduzidas , Animais , Linfócitos B/metabolismo , Sequência de Bases , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células-Tronco Embrionárias , Fator de Iniciação 4E em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células NIH 3T3 , Ribose-Fosfato Pirofosfoquinase/metabolismoRESUMO
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.
Assuntos
Ambystoma mexicanum , Evolução Biológica , Biossíntese de Proteínas , Regeneração , Serina-Treonina Quinases TOR , Animais , Humanos , Camundongos , Ambystoma mexicanum/fisiologia , Sequência de Aminoácidos , Extremidades/fisiologia , Regeneração/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Cicatrização , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Especificidade da Espécie , Antioxidantes/metabolismo , Nutrientes/metabolismo , Polirribossomos/genética , Polirribossomos/metabolismoRESUMO
Post-transcriptional regulation of RNA stability is a key step in gene expression control. We describe a regulatory program, mediated by the RNA binding protein TARBP2, that controls RNA stability in the nucleus. TARBP2 binding to pre-mRNAs results in increased intron retention, subsequently leading to targeted degradation of TARBP2-bound transcripts. This is mediated by TARBP2 recruitment of the m6A RNA methylation machinery to its target transcripts, where deposition of m6A marks influences the recruitment of splicing regulators, inhibiting efficient splicing. Interactions between TARBP2 and the nucleoprotein TPR then promote degradation of these TARBP2-bound transcripts by the nuclear exosome. Additionally, analysis of clinical gene expression datasets revealed a functional role for TARBP2 in lung cancer. Using xenograft mouse models, we find that TARBP2 affects tumor growth in the lung and that this is dependent on TARBP2-mediated destabilization of ABCA3 and FOXN3. Finally, we establish ZNF143 as an upstream regulator of TARBP2 expression.
Assuntos
Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Translational control of mRNAs is a point of convergence for many oncogenic signals through which cancer cells tune protein expression in tumorigenesis. Cancer cells rely on translational control to appropriately adapt to limited resources while maintaining cell growth and survival, which creates a selective therapeutic window compared to non-transformed cells. In this review, we first discuss how cancer cells modulate the translational machinery to rapidly and selectively synthesize proteins in response to internal oncogenic demands and external factors in the tumor microenvironment. We highlight the clinical potential of compounds that target different translation factors as anti-cancer therapies. Next, we detail how RNA sequence and structural elements interface with the translational machinery and RNA-binding proteins to coordinate the translation of specific pro-survival and pro-growth programs. Finally, we provide an overview of the current and emerging technologies that can be used to illuminate the mechanisms of selective translational control in cancer cells as well as within the microenvironment.
Assuntos
Neoplasias , Biossíntese de Proteínas , Carcinogênese , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , RNA Mensageiro/metabolismo , Microambiente TumoralRESUMO
In this issue of Molecular Cell, Tebaldi et al. (2018) identify the neuron-specific RNA-binding protein HuD as a regulator of global protein synthesis and translation enhancer of specific mTORC1-responsive transcripts. Importantly, the authors identify that the Y3 small non-coding RNA binds HuD to modulate translation and neurogenesis.
Assuntos
Proteína Semelhante a ELAV 4 , Alvo Mecanístico do Complexo 1 de Rapamicina , Neurônios , RNA Mensageiro , Pequeno RNA não Traduzido , Proteínas de Ligação a RNARESUMO
Aberrantly high mTORC1 signaling is a known driver of many cancers and human disorders, yet pharmacological inhibition of mTORC1 rarely confers durable clinical responses. To explore alternative therapeutic strategies, herein we conducted a proteomics survey to identify cell surface proteins upregulated by mTORC1. A comparison of the surfaceome from Tsc1-/-versus Tsc1+/+ mouse embryonic fibroblasts revealed 59 proteins predicted to be significantly overexpressed in Tsc1-/- cells. Further validation of the data in multiple mouse and human cell lines showed that mTORC1 signaling most dramatically induced the expression of the proteases neprilysin (NEP/CD10) and aminopeptidase N (APN/CD13). Functional studies showed that constitutive mTORC1 signaling sensitized cells to genetic ablation of NEP and APN, as well as the biochemical inhibition of APN. In summary, these data show that mTORC1 signaling plays a significant role in the constitution of the surfaceome, which in turn may present novel therapeutic strategies.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Animais , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/genética , Antígenos CD13/metabolismo , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neprilisina/genética , Neprilisina/metabolismo , Proteômica , RNA Interferente Pequeno , Transdução de Sinais , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
During an adaptive immune response, activated mature B cells give rise to Ab-secreting plasma cells to fight infection. B cells undergo Ab class switching to produce different classes of Abs with varying effector functions. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is activated during this process, and disrupting mTOR complex 1 (mTORC1) in B cells impairs class switching by a poorly understood mechanism. In particular, it is unclear which mTORC1 downstream substrates control this process. In this study, we used an in vitro murine model in which the mTORC1 inhibitor rapamycin, when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Investigation of mTORC1 substrates revealed a role for eukaryotic translation initiation factor 4E (eIF4E) and eIF4E-binding proteins in class switching. Mechanistically, we show that genetic or pharmacological disruption of eIF4E binding to eIF4G reduced cap-dependent translation, which specifically affected the expression of activation-induced cytidine deaminase protein but not Aicda mRNA. This translational impairment decreased Ab class switching independently of proliferation. These results uncover a previously undescribed role for mTORC1 and the eIF4E-binding proteins/eIF4E axis in activation-induced cytidine deaminase protein expression and Ab class switching in mouse B cells, suggesting that cap-dependent translation regulates key steps in B cell differentiation.
Assuntos
Linfócitos B/imunologia , Proteínas de Transporte/imunologia , Fator de Iniciação 4E em Eucariotos/imunologia , Switching de Imunoglobulina , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Fosfoproteínas/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/genética , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/genética , Ligação Proteica , Biossíntese de Proteínas , Transdução de Sinais , Sirolimo/farmacologiaRESUMO
Gene regulation during cell-cycle progression is an intricately choreographed process, ensuring accurate DNA replication and division. However, the translational landscape of gene expression underlying cell-cycle progression remains largely unknown. Employing genome-wide ribosome profiling, we uncover widespread translational regulation of hundreds of mRNAs serving as an unexpected mechanism for gene regulation underlying cell-cycle progression. A striking example is the S phase translational regulation of RICTOR, which is associated with cell cycle-dependent activation of mammalian target of rapamycin complex 2 (mTORC2) signaling and accurate cell-cycle progression. We further identified unappreciated coordination in translational control of mRNAs within molecular complexes dedicated to cell-cycle progression, lipid metabolism, and genome integrity. This includes the majority of mRNAs comprising the cohesin and condensin complexes responsible for maintaining genome organization, which are coordinately translated during specific cell cycle phases via their 5' UTRs. Our findings illuminate the prevalence and dynamic nature of translational regulation underlying the mammalian cell cycle.
Assuntos
Regulação da Expressão Gênica , Mitose/genética , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Transporte Ativo do Núcleo Celular/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Ciclo do Ácido Cítrico/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Genoma Humano , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma , CoesinasRESUMO
Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography-tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.
Assuntos
Diferenciação Celular/fisiologia , Proteoma/análise , Proteômica/métodos , Transdução de Sinais/fisiologia , Cromatografia Líquida , Descoberta de Drogas , Humanos , Células K562 , Biossíntese de Proteínas , Proteoma/química , Proteoma/metabolismo , Puromicina/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Espectrometria de Massas em TandemRESUMO
Autism spectrum disorders (ASDs) are an early onset, heterogeneous group of heritable neuropsychiatric disorders with symptoms that include deficits in social interaction skills, impaired communication abilities, and ritualistic-like repetitive behaviours. One of the hypotheses for a common molecular mechanism underlying ASDs is altered translational control resulting in exaggerated protein synthesis. Genetic variants in chromosome 4q, which contains the EIF4E locus, have been described in patients with autism. Importantly, a rare single nucleotide polymorphism has been identified in autism that is associated with increased promoter activity in the EIF4E gene. Here we show that genetically increasing the levels of eukaryotic translation initiation factor 4E (eIF4E) in mice results in exaggerated cap-dependent translation and aberrant behaviours reminiscent of autism, including repetitive and perseverative behaviours and social interaction deficits. Moreover, these autistic-like behaviours are accompanied by synaptic pathophysiology in the medial prefrontal cortex, striatum and hippocampus. The autistic-like behaviours displayed by the eIF4E-transgenic mice are corrected by intracerebroventricular infusions of the cap-dependent translation inhibitor 4EGI-1. Our findings demonstrate a causal relationship between exaggerated cap-dependent translation, synaptic dysfunction and aberrant behaviours associated with autism.
Assuntos
Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Biossíntese de Proteínas , Sinapses/metabolismo , Sinapses/patologia , Animais , Transtorno Autístico/tratamento farmacológico , Transtorno Autístico/patologia , Comportamento Animal/efeitos dos fármacos , Dendritos/metabolismo , Dendritos/patologia , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Feminino , Hipocampo/metabolismo , Hidrazonas , Infusões Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Neostriado/metabolismo , Plasticidade Neuronal , Nitrocompostos/administração & dosagem , Nitrocompostos/farmacologia , Nitrocompostos/uso terapêutico , Córtex Pré-Frontal/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Capuzes de RNA/metabolismo , Tiazóis/administração & dosagem , Tiazóis/farmacologia , Tiazóis/uso terapêuticoRESUMO
Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.
Assuntos
Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Biossíntese de Proteínas , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/deficiência , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Sinapses/metabolismoRESUMO
How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.
Assuntos
Proteínas de Ciclo Celular/deficiência , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Hidroliases/deficiência , Hidroliases/metabolismo , Deficiência Intelectual/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Nucleares/deficiência , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas Nucleares Pequenas/deficiência , Saccharomyces cerevisiae/genética , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Disceratose Congênita/enzimologia , Retardo do Crescimento Fetal/enzimologia , Genes Reporter , Humanos , Hidroliases/genética , Deficiência Intelectual/enzimologia , Luciferases/análise , Camundongos , Microcefalia/enzimologia , Proteínas Associadas aos Microtúbulos/genética , Mutação , Proteínas Nucleares/genética , Plasmídeos , Biossíntese de Proteínas , RNA Ribossômico/química , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transdução GenéticaRESUMO
The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the 'cancerous' translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.