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1.
Dev Cell ; 56(18): 2607-2622.e6, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34534458

RESUMO

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.

2.
Nat Commun ; 12(1): 4878, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385447

RESUMO

A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPß, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Hepatopatia Gordurosa não Alcoólica/genética , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Clofazimina/farmacologia , Dieta Hiperlipídica/efeitos adversos , Progressão da Doença , Inativação Gênica , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Obesidade/genética , Obesidade/metabolismo , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/metabolismo
3.
Cancer Res ; 80(21): 4693-4706, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32963011

RESUMO

Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is FAM46C that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.


Assuntos
Fibronectinas/metabolismo , Mieloma Múltiplo/patologia , Nucleotidiltransferases/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Autofagia/fisiologia , Genes Supressores de Tumor , Xenoenxertos , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Nucleotidiltransferases/genética , Agregados Proteicos/fisiologia , Transporte Proteico/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
4.
Dev Comp Immunol ; 109: 103697, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32330465

RESUMO

The transition from a naïve to an effector T cell is an essential event that requires metabolic reprogramming. We have recently demonstrated that the rapid metabolic changes that occur following stimulation of naïve T cells require the translation of preexisting mRNAs. Here, we provide evidence that translation regulates the metabolic asset of effector T cells. By performing ribosome profiling in human CD4+ Th1 cells, we show that the metabolism of glucose, fatty acids and pentose phosphates is regulated at the translational level. In Th1 cells, each pathway has at least one enzyme regulated at the translational level and selected enzymes have high translational efficiencies. mRNA expression does not predict protein expression. For instance, PKM2 mRNA is equally present in naïve T and Th1 cells, but the protein is abundant only in Th1. 5'-untranslated regions (UTRs) may partly account for this regulation. Overall we suggest that immunometabolism is controlled by translation.

5.
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702

RESUMO

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
7.
Cell Signal ; 53: 102-110, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30287278

RESUMO

Neuroblastoma is the most frequent solid tumor among those diagnosed during infancy and like most tumors, it is characterized by elevated rates of cell proliferation, migration and invasion. RACK1 is among the top 10 genes identified for unfavorable prognosis at 5 years in neuroblastoma cases and its depletion negatively affects proliferation, invasion and migration. Here, we show that the ribosomal localization of RACK1 modulates the proliferation of SH-SY5Y neuroblastoma cells by regulating the expression of cell cycle genes, such as Cyclin D1, D3 and B1 independently of global translation increase. Ribosomal RACK1 is not involved in general protein synthesis, which is instead dependent on total RACK1 and PKC but independent from mTOR. Thus, ribosomal RACK1 may represent a new target to develop more efficient therapies for neuroblastoma treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Biossíntese de Proteínas , Receptores de Quinase C Ativada/genética , Ribossomos/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos
9.
Cell Metab ; 28(6): 895-906.e5, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30197303

RESUMO

Naive T cells respond to T cell receptor (TCR) activation by leaving quiescence, remodeling metabolism, initiating expansion, and differentiating toward effector T cells. The molecular mechanisms coordinating the naive to effector transition are central to the functioning of the immune system, but remain elusive. Here, we discover that T cells fulfill this transitional process through translational control. Naive cells accumulate untranslated mRNAs encoding for glycolysis and fatty acid synthesis factors and possess a translational machinery poised for immediate protein synthesis. Upon TCR engagement, activation of the translational machinery leads to synthesis of GLUT1 protein to drive glucose entry. Subsequently, translation of ACC1 mRNA completes metabolic reprogramming toward an effector phenotype. Notably, inhibition of the eIF4F complex abrogates lymphocyte metabolic activation and differentiation, suggesting ACC1 to be a key regulatory node. Thus, our results demonstrate that translation is a direct mediator of T cell metabolism and indicate translation factors as targets for novel immunotherapeutic approaches.


Assuntos
Acetil-CoA Carboxilase/biossíntese , Linfócitos T CD4-Positivos/metabolismo , Ácidos Graxos/metabolismo , Transportador de Glucose Tipo 1/biossíntese , Glicólise , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Proliferação de Células , Fator de Iniciação 4F em Eucariotos/antagonistas & inibidores , Humanos , Ativação Linfocitária , Biossíntese de Proteínas , Subpopulações de Linfócitos T/citologia
10.
Mol Cell Biol ; 38(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30201806

RESUMO

The translational capability of ribosomes deprived of specific nonfundamental ribosomal proteins may be altered. Physiological mechanisms are scanty, and it is unclear whether free ribosomal proteins can cross talk with the signaling machinery. RACK1 (receptor for activated C kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling, such as the protein kinase C (PKC) family. Here we show that the binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eukaryotic initiation factor 4E (eIF4E). In vitro, when RACK1 is partially depleted, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas the addition of a RACK1 mutant that is unable to bind ribosomes does not. Outside the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype, impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly through interaction with known partners on or off the ribosome that are involved in signaling.

11.
Curr Opin Genet Dev ; 48: 75-81, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29153483

RESUMO

Albeit cancer patients' heterogeneity, all tumor cells have alterations of both metabolism and translation. The simplest explanation for this common feature is that several oncogenes coordinate a translational and metabolic reprogramming that is necessary for tumor cells to thrive. Overall, at least three oncogenic pathways, namely c-Myc, RAS and PI3K-mTOR, are known to affect both translation and metabolism by stimulating glycolysis and protein synthesis. The crosstalk between metabolite production and the translational machinery is, instead, less understood. What is known is that, on one side, translation initiation factors, such as eIF4E and eIF6, drive tumor growth and regulate metabolism through selective translation of nucleotide biosynthesis, glycolysis and fatty acid synthesis rate-limiting mRNAs, and on the other, that nutrient levels regulate the translational machinery by inducing full activity of translation factors. Therefore, translation and metabolism offer several therapeutic targets to be fully exploited in future studies.


Assuntos
Neoplasias/metabolismo , Biossíntese de Proteínas , Animais , Carcinogênese , Humanos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo
12.
Data Brief ; 14: 653-658, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28924581

RESUMO

The data described in this article are related to "High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans" (Manfrini et al., in press) [1]. eIF6 is a translation initiation factor required for ribosomal biogenesis (Sanvito et al., 1999) [2] and for proper translational initiation (Gallo and Manfrini, 2015; Miluzio et al., 2016) [3], [4] whose protein abundance requires tight regulation. Here we analyze by flow cytometry the effects of eIF6 depletion on proportions of specific innate and adaptive immune system subpopulations and on thymocyte maturation in mice.

13.
Dev Comp Immunol ; 77: 69-76, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28743432

RESUMO

Eukaryotic Initiation Factor 6 (eIF6) is required for 60S ribosomal subunit biogenesis and efficient initiation of translation. Intriguingly, in both mice and humans, endogenous levels of eIF6 are detrimental as they act as tumor and obesity facilitators, raising the question on the evolutionary pressure that maintains high eIF6 levels. Here we show that, in mice and humans, high levels of eIF6 are required for proper immune functions. First, eIF6 heterozygous (het) mice show an increased mortality during viral infection and a reduction of peripheral blood CD4+ Effector Memory T cells. In human CD4+ T cells, eIF6 levels rapidly increase upon T-cell receptor activation and drive the glycolytic switch and the acquisition of effector functions. Importantly, in CD4+ T cells, eIF6 levels control interferon-γ (IFN-γ) secretion without affecting proliferation. In conclusion, the immune system has a high evolutionary pressure for the maintenance of a dynamic and powerful regulation of the translational machinery.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Viroses/imunologia , Animais , Células Cultivadas , Fatores de Iniciação em Eucariotos/genética , Glicólise , Homeostase , Humanos , Sistema Imunitário , Memória Imunológica , Interferon gama/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Iniciação de Peptídeos/genética , Transdução de Sinais
14.
World J Biol Chem ; 8(1): 45-56, 2017 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-28289518

RESUMO

MicroRNAs (miRNAs) are pervasively expressed and regulate most biological functions. They function by modulating transcriptional and translational programs and therefore they orchestrate both physiological and pathological processes, such as development, cell differentiation, proliferation, apoptosis and tumor growth. miRNAs work as small guide molecules in RNA silencing, by negatively regulating the expression of several genes both at mRNA and protein level, by degrading their mRNA target and/or by silencing translation. One of the most recent advances in the field is the comprehension of their role in oncogenesis. The number of miRNA genes is increasing and an alteration in the level of miRNAs is involved in the initiation, progression and metastases formation of several tumors. Some tumor types show a distinct miRNA signature that distinguishes them from normal tissues and from other cancer types. Genetic and biochemical evidence supports the essential role of miRNAs in tumor development. Although the abnormal expression of miRNAs in cancer cells is a widely accepted phenomenon, the cause of this dysregulation is still unknown. Here, we discuss the biogenesis of miRNAs, focusing on the mechanisms by which they regulate protein synthesis. In addition we debate on their role in cancer, highlighting their potential to become therapeutic targets.

15.
Biochem Soc Trans ; 44(6): 1667-1673, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27913676

RESUMO

Over the past few years, there has been a growing interest in the interconnection between translation and metabolism. Important oncogenic pathways, like those elicited by c-Myc transcription factor and mTOR kinase, couple the activation of the translational machinery with glycolysis and fatty acid synthesis. Eukaryotic initiation factor 6 (eIF6) is a factor necessary for 60S ribosome maturation. eIF6 acts also as a cytoplasmic translation initiation factor, downstream of growth factor stimulation. eIF6 is up-regulated in several tumor types. Data on mice models have demonstrated that eIF6 cytoplasmic activity is rate-limiting for Myc-induced lymphomagenesis. In spite of this, eIF6 is neither transcriptionally regulated by Myc, nor post-transcriptionally regulated by mTOR. eIF6 stimulates a glycolytic and fatty acid synthesis program necessary for tumor growth. eIF6 increases the translation of transcription factors necessary for lipogenesis, such as CEBP/ß, ATF4 and CEBP/δ. Insulin stimulation leads to an increase in translation and fat synthesis blunted by eIF6 deficiency. Paradoxycally, long-term inhibition of eIF6 activity increases insulin sensitivity, suggesting that the translational activation observed upon insulin and growth factors stimulation acts as a feed-forward mechanism regulating lipid synthesis. The data on the role that eIF6 plays in cancer and in insulin sensitivity make it a tempting pharmacological target for cancers and metabolic diseases. We speculate that eIF6 inhibition will be particularly effective especially when mTOR sensitivity to rapamycin is abrogated by RAS mutations.


Assuntos
Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Camundongos , Modelos Genéticos , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo
16.
Elife ; 42015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26231041

RESUMO

Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism. Here we show that in Saccharomyces cerevisiae a single DSB causes transcriptional inhibition of proximal genes independently of Tel1/ATM and Mec1/ATR. Since the DSB ends undergo nucleolytic degradation (resection) of their 5'-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition. We discovered that resection-defective mutants fail to stop transcription around a DSB, and the extent of this failure correlates with the severity of the resection defect. Furthermore, Rad9 and generation of γH2A reduce this DSB-induced transcriptional inhibition by counteracting DSB resection. Therefore, the conversion of the DSB ends from double-stranded to single-stranded DNA, which is necessary to initiate DSB repair by homologous recombination, is responsible for loss of transcription around a DSB in S. cerevisiae.


Assuntos
Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Genética
17.
Translation (Austin) ; 3(2): e1120382, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26824030

RESUMO

RACK1 is a ribosome-associated protein which functions as a receptor for activated PKCs. It also acts as a scaffold for many other proteins involved in diverse signaling pathways, e.g. Src, JNK, PDE4D and FAK signaling. With such a broad interactome, RACK1 has been suggested to function as a linker between cell signaling and the translation machinery. Accordingly, RACK1 modulates translation at different levels in several model organisms. For instance, it regulates ribosome stalling and mRNA quality control in yeasts and promotes translation efficiency downstream of specific cellular stimuli in mammals. However, the molecular mechanism by which RACK1 exerts these roles is widely uncharacterized. Moreover, the full list of ribosome-recruited RACK1 interactors still needs characterization. Here we discuss in vivo and in vitro findings to better delineate the roles of RACK1 in regulating ribosome function and translation.

18.
EMBO Rep ; 16(2): 221-31, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25527408

RESUMO

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.


Assuntos
DNA de Cadeia Simples/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
J Mol Biol ; 425(23): 4756-66, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-23603016

RESUMO

The eukaryotic cell cycle comprises a series of events, whose ordering and correct progression depends on the oscillating activity of cyclin-dependent kinases (Cdks), which safeguard timely duplication and segregation of the genome. Cell division is intimately connected to an evolutionarily conserved DNA damage response (DDR), which involves DNA repair pathways that reverse DNA lesions, as well as checkpoint pathways that inhibit cell cycle progression while repair occurs. There is increasing evidence that Cdks are involved in the DDR, in particular in DNA repair by homologous recombination and in activation of the checkpoint response. However, Cdks have to be carefully regulated, because even an excess of their activity can affect genome stability. In this review, we consider the physiological role of Cdks in the DDR.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Reparo do DNA , Eucariotos/genética , Eucariotos/fisiologia , Regulação da Expressão Gênica
20.
Mol Cell Biol ; 32(24): 4971-85, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23045388

RESUMO

The Mec1 and Rad53 protein kinases are essential for budding yeast cell viability and are also required to activate the S-phase checkpoint, which supports DNA replication under stress conditions. Whether these two functions are related to each other remains to be determined, and the nature of the replication stress-dependent lethality of mec1 and rad53 mutants is still unclear. We show here that a decrease in cyclin-dependent kinase 1 (Cdk1) activity alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of a certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G(1) and mitosis. In fact, delaying either the G(1)/S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to hydroxyurea and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions.


Assuntos
Proteína Quinase CDC2/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Replicação do DNA , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Genes Fúngicos , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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