RESUMO
Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory capacity in mRNA translation. Here we identify mutations of the Ribosomal Protein L38 (Rpl38) gene in mice exhibiting surprising tissue-specific patterning defects, including pronounced homeotic transformations of the axial skeleton. In Rpl38 mutant embryos, global protein synthesis is unchanged; however the translation of a select subset of Homeobox mRNAs is perturbed. Our data reveal that RPL38 facilitates 80S complex formation on these mRNAs as a regulatory component of the ribosome to confer transcript-specific translational control. We further show that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes occur. Unexpectedly, a ribosomal protein (RP) expression screen reveals dynamic regulation of individual RPs within the vertebrate embryo. Collectively, these findings suggest that RP activity may be highly regulated to impart a new layer of specificity in the control of gene expression and mammalian development.
Assuntos
Padronização Corporal , Doenças do Desenvolvimento Ósseo/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Organogênese , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Cauda/anormalidadesRESUMO
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
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.
Assuntos
Metástase Neoplásica , Neoplasias da Próstata/patologia , Biossíntese de Proteínas , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Benzoxazóis/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genoma/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/genética , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/genética , Fosfoproteínas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
MOTIVATION: The translational landscape of diverse cellular systems remains largely uncharacterized. A detailed understanding of the control of gene expression at the level of messenger RNA translation is vital to elucidating a systems-level view of complex molecular programs in the cell. Establishing the degree to which such post-transcriptional regulation can mediate specific phenotypes is similarly critical to elucidating the molecular pathogenesis of diseases such as cancer. Recently, methods for massively parallel sequencing of ribosome-bound fragments of messenger RNA have begun to uncover genome-wide translational control at codon resolution. Despite its promise for deeply characterizing mammalian proteomes, few analytical methods exist for the comprehensive analysis of this paired RNA and ribosome data. RESULTS: We describe the Babel framework, an analytical methodology for assessing the significance of changes in translational regulation within cells and between conditions. This approach facilitates the analysis of translation genome-wide while allowing statistically principled gene-level inference. Babel is based on an errors-in-variables regression model that uses the negative binomial distribution and draws inference using a parametric bootstrap approach. We demonstrate the operating characteristics of Babel on simulated data and use its gene-level inference to extend prior analyses significantly, discovering new translationally regulated modules under mammalian target of rapamycin (mTOR) pathway signaling control.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Software , Algoritmos , Animais , Códon/metabolismo , Simulação por Computador , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/genética , Ribossomos/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.
RESUMO
Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extra-terminal (BET) protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent anti-tumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent anti-tumor activity in vivo.
RESUMO
A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the "core" PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.
Assuntos
Mitocôndrias/metabolismo , Modelos Moleculares , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Cristalografia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/genética , Oligonucleotídeos/genética , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Oncoprotein expression is controlled at the level of mRNA translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, a component of eIF4F, catalyzes the unwinding of secondary structure in the 5'-untranslated region (5'-UTR) of mRNA to facilitate ribosome scanning and translation initiation. Zotatifin (eFT226) is a selective eIF4A inhibitor that increases the affinity between eIF4A and specific polypurine sequence motifs and has been reported to inhibit translation of driver oncogenes in models of lymphoma. Here we report the identification of zotatifin binding motifs in the 5'-UTRs of HER2 and FGFR1/2 Receptor Tyrosine Kinases (RTKs). Dysregulation of HER2 or FGFR1/2 in human cancers leads to activation of the PI3K/AKT and RAS/ERK signaling pathways, thus enhancing eIF4A activity and promoting the translation of select oncogenes that are required for tumor cell growth and survival. In solid tumor models driven by alterations in HER2 or FGFR1/2, downregulation of oncoprotein expression by zotatifin induces sustained pathway-dependent anti-tumor activity resulting in potent inhibition of cell proliferation, induction of apoptosis, and significant in vivo tumor growth inhibition or regression. Sensitivity of RTK-driven tumor models to zotatifin correlated with high basal levels of mTOR activity and elevated translational capacity highlighting the unique circuitry generated by the RTK-driven signaling pathway. This dependency identifies the potential for rational combination strategies aimed at vertical inhibition of the PI3K/AKT/eIF4F pathway. Combination of zotatifin with PI3K or AKT inhibitors was beneficial across RTK-driven cancer models by blocking RTK-driven resistance mechanisms demonstrating the clinical potential of these combination strategies.
RESUMO
Fragile X syndrome (FXS) is the most common inherited source of intellectual disability in humans. FXS is caused by mutations that trigger epigenetic silencing of the Fmr1 gene. Loss of Fmr1 results in increased activity of the mitogen-activated protein kinase (MAPK) pathway. An important downstream consequence is activation of the mitogen-activated protein kinase interacting protein kinase (MNK). MNK phosphorylates the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). Excessive phosphorylation of eIF4E has been directly implicated in the cognitive and behavioral deficits associated with FXS. Pharmacological reduction of eIF4E phosphorylation is one potential strategy for FXS treatment. We demonstrate that systemic dosing of a highly specific, orally available MNK inhibitor, eFT508, attenuates numerous deficits associated with loss of Fmr1 in mice. eFT508 resolves a range of phenotypic abnormalities associated with FXS including macroorchidism, aberrant spinogenesis, and alterations in synaptic plasticity. Key behavioral deficits related to anxiety, social interaction, obsessive and repetitive activities, and object recognition are ameliorated by eFT508. Collectively, this work establishes eFT508 as a potential means to reverse deficits associated with FXS.
Assuntos
Síndrome do Cromossomo X Frágil/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Síndrome do Cromossomo X Frágil/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Teste de Campo Aberto/efeitos dos fármacos , Comportamento SocialRESUMO
PUF proteins comprise a highly conserved family of sequence-specific RNA binding proteins that regulate target mRNAs via binding directly to their 3'UTRs. The Caenorhabditis elegans genome encodes several PUF proteins, which cluster into four groups based on sequence similarity; all share amino acids that interact with the RNA in the cocrystal of human Pumilio with RNA. Members of the FBF and the PUF-8/9 groups bind different but related RNA sequences. We focus here on the binding specificity of representatives of a third cluster, comprising PUF-5, -6, and -7. We performed in vivo selection experiments using the yeast three-hybrid system to identify RNA sequences that bind PUF-5 and PUF-6, and we confirmed binding to optimal sites in vitro. The consensus sequences derived from the screens are similar for PUF-5 and PUF-6 but differ from those of the FBF or PUF-8/-9 groups. Similarly, neither PUF-5 nor PUF-6 bind the recognition sites preferred by the other clusters. Mutagenesis studies confirmed the unique RNA specificity of PUF-5/-6. Using the PUF-5 consensus derived from our experiments, we searched a database of C. elegans 3'UTRs to identify potential targets of PUF-5, several of which indeed bind PUF-5. Therefore the consensus has predictive value and provides a route to finding genuine targets of these proteins.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Família Multigênica , Proteínas de Ligação a RNA/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.
Assuntos
Imunoterapia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Biossíntese de Proteínas , Regiões 5' não Traduzidas/genética , Animais , Antígeno B7-H1/metabolismo , Sequência de Bases , Progressão da Doença , Regulação para Baixo , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Evasão da Resposta Imune , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Fases de Leitura Aberta/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Transcrição Gênica , Microambiente Tumoral , Regulação para Cima/genéticaRESUMO
Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.
Assuntos
Antineoplásicos/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/uso terapêutico , Piridonas/uso terapêutico , Pirimidinas/uso terapêutico , Compostos de Espiro/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Estrutura Molecular , Fosforilação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Piridonas/síntese química , Piridonas/química , Piridonas/farmacologia , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Serina/química , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/síntese química , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is an increasing realization that a primary role for Myc in driving cellular growth and cell cycle progression relies on Myc's ability to increase the rate of protein synthesis. Myc induces myriad changes in both global and specific mRNA translation. Herein, we present three assays that allow researchers to measure changes in protein synthesis at the global level as well as alterations in the translation of specific mRNAs. Metabolic labeling of cells with (35)S-containing methionine and cysteine is presented as a method to measure the overall rate of global protein synthesis. The bicistronic reporter assay is employed to determine levels of cap-dependent and cap-independent translation initiation in the cell. Finally, isolation of polysome-associated mRNAs followed by next-generation sequencing, microarray or quantitative real-time PCR (qRT-PCR) analysis is utilized to detect changes in the abundance of specific mRNAs that are regulated upon Myc hyperactivation. The protocols described in this chapter can be used to understand how and to what extent Myc-dependent regulation of translation influences normal cellular functions as well as tumorigenesis.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Humanos , Iniciação Traducional da Cadeia Peptídica , Capuzes de RNA/genética , Capuzes de RNA/metabolismoRESUMO
Deregulations in translational control are critical features of cancer initiation and progression. Activation of key oncogenic pathways promotes rapid and dramatic translational reprogramming, not simply by increasing overall protein synthesis, but also by modulating specific mRNA networks that promote cellular transformation. Additionally, ribosomopathies caused by mutations in ribosome components alter translational regulation leading to specific pathological features, including cancer susceptibility. Exciting advances in our understanding of translational control in cancer have illuminated a striking specificity innate to the translational apparatus. Characterizing this specificity will provide novel insights into how cells normally utilize translational control to modulate gene expression, how it is deregulated in cancer, and how these processes can be targeted to develop new cancer therapies.
Assuntos
Predisposição Genética para Doença , Neoplasias/genética , Neoplasias/patologia , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
RNA-protein interactions play an essential role in the maturation and regulation of RNAs within eukaryotic organisms. The three-hybrid system provides a simple, yet powerful means to study RNA-protein interactions within the eukaryote Saccharomyces cerevisiae. This chapter describes the basis of the system and applications in both examining specific RNA-protein interactions and screening libraries for novel interactions. We provide a detailed discussion on affinity versus reporter output, variations on library screening (e.g., randomization studies), some adaptations of the system, and updated reagents and protocols.