RESUMO
The transcription factor p53 exerts its tumour suppressive effect through transcriptional activation of numerous target genes controlling cell cycle arrest, apoptosis, cellular senescence and DNA repair. In addition, there is evidence that p53 influences the translation of specific mRNAs, including translational inhibition of ribosomal protein synthesis and translational activation of MDM2. A challenge in the analysis of translational control is that changes in mRNA abundance exert a kinetic (passive) effect on ribosome densities. In order to separate these passive effects from active regulation of translation efficiency in response to p53 activation, we conducted a comprehensive analysis of translational regulation by comparative analysis of mRNA levels and ribosome densities upon DNA damage induced by neocarzinostatin in wild-type and TP53-/- HCT116 colorectal carcinoma cells. Thereby, we identified a specific group of mRNAs that are preferentially translated in response to p53 activation, many of which correspond to p53 target genes including MDM2, SESN1 and CDKN1A. By subsequent polysome profile analysis of SESN1 and CDKN1A mRNA, we could demonstrate that p53-dependent translational activation relies on a combination of inducing the expression of translationally advantageous isoforms and trans-acting mechanisms that further enhance the translation of these mRNAs.
Assuntos
Ribossomos , Proteína Supressora de Tumor p53 , Pontos de Checagem do Ciclo Celular , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The CCR4-NOT complex acts as a central player in the control of mRNA turnover and mediates accelerated mRNA degradation upon HDAC inhibition. Here, we explored acetylation-induced changes in the composition of the CCR4-NOT complex by purification of the endogenously tagged scaffold subunit NOT1 and identified RNF219 as an acetylation-regulated cofactor. We demonstrate that RNF219 is an active RING-type E3 ligase which stably associates with CCR4-NOT via NOT9 through a short linear motif (SLiM) embedded within the C-terminal low-complexity region of RNF219. By using a reconstituted six-subunit human CCR4-NOT complex, we demonstrate that RNF219 inhibits deadenylation through the direct interaction of the α-helical SLiM with the NOT9 module. Transcriptome-wide mRNA half-life measurements reveal that RNF219 attenuates global mRNA turnover in cells, with differential requirement of its RING domain. Our results establish RNF219 as an inhibitor of CCR4-NOT-mediated deadenylation, whose loss upon HDAC inhibition contributes to accelerated mRNA turnover.
Assuntos
RNA Mensageiro/metabolismo , Receptores CCR4/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Monofosfato de Adenosina/metabolismo , Células HeLa , Humanos , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Receptores CCR4/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.
Assuntos
Proteína Quinase CDC2/metabolismo , Proliferação de Células , Neoplasias do Colo do Útero/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinética , Camundongos Endogâmicos C57BL , Fosforilação , Biossíntese de Proteínas , Sequência de Oligopirimidina na Região 5' Terminal do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Antígeno SS-BRESUMO
Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the ß-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.
Assuntos
Proteínas de Drosophila/química , Histona-Lisina N-Metiltransferase/química , Histonas/química , Proteínas Repressoras/química , Proteína 4 de Ligação ao Retinoblastoma/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Estrutura Secundária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismoRESUMO
Polycomb group (PcG) proteins repress transcription by modifying chromatin structure in target genes. dSfmbt is a subunit of the Drosophila melanogaster PcG protein complex PhoRC and contains four malignant brain tumour (MBT) repeats involved in the recognition of various mono- and dimethylated histone peptides. Here, we present the crystal structure of the four-MBT-repeat domain of dSfmbt in complex with a mono-methylated histone H4 peptide. Only a single histone peptide binds to the four-MBT-repeat domain. Mutational analyses show high-affinity binding with low peptide sequence selectivity through combinatorial interaction of the methyl-lysine with an aromatic cage and positively charged flanking residues with the surrounding negatively charged surface of the fourth MBT repeat. dSfmbt directly interacts with the PcG protein Scm, a related MBT-repeat protein with similar methyl-lysine binding activity. dSfmbt and Scm co-occupy Polycomb response elements of target genes in Drosophila and they strongly synergize in the repression of these target genes, suggesting that the combined action of these two MBT proteins is crucial for Polycomb silencing.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histonas/genética , Humanos , Lisina/química , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Alinhamento de SequênciaRESUMO
The chromosomal passenger complex (CPC) is a key regulator of chromosome segregation and cytokinesis. CPC functions are connected to its localization. The complex first localizes to centromeres and later associates with the central spindle and midbody. Survivin, Borealin, and INCENP are the three components of the CPC that regulate the activity and localization of its enzymatic component, the kinase Aurora B. We determined the 1.4 A resolution crystal structure of the regulatory core of the CPC, revealing that Borealin and INCENP associate with the helical domain of Survivin to form a tight three-helical bundle. We used siRNA rescue experiments with structure-based mutants to explore the requirements for CPC localization. We show that the intertwined structural interactions of the core components lead to functional interdependence. Association of the core "passenger" proteins creates a single structural unit, whose composite molecular surface presents conserved residues essential for central spindle and midbody localization.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos/fisiologia , Proteínas Associadas aos Microtúbulos/química , Proteínas de Neoplasias/química , Fuso Acromático/fisiologia , Sequência de Aminoácidos , Aurora Quinase B , Aurora Quinases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Centrômero/genética , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Segregação de Cromossomos/genética , Citocinese , Dimerização , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fuso Acromático/genética , SurvivinaRESUMO
The mitotic kinases Aurora-A and Aurora-B have similar amino-acid sequences but are differently localised and regulated during cell division. The basis for their interactions with different and specific regulators is unclear. Surprisingly, our recent structural studies indicate that TPX2 regulates Aurora-A activity by binding at a site that is conserved almost completely on Aurora-B. Here we investigate molecular determinants of TPX2-Aurora-A recognition. Using structure-based mutagenesis, we show that a single amino-acid difference on the surface of the kinase catalytic domain is key to the precision with which TPX2 discriminates between Aurora-A and Aurora-B. The conservation at this amino-acid position suggests that this discriminatory mechanism is likely to be conserved in higher eukaryotes.