RESUMO
Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, a specific activator of cyclin-dependent kinase 5 (CDK5). CDK5 and p35 have a fundamental role in neuronal migration and differentiation during CNS development. Both the CDK5R1 3'-UTR's remarkable size and its conservation during evolution strongly indicate an important role in post-transcriptional regulation. We previously validated different regulatory elements in the 3'-UTR of CDK5R1, which affect transcript stability, p35 levels and cellular migration through the binding with nELAV proteins and miR-103/7 miRNAs. Interestingly, a 138 bp-long region, named C2.1, was identified as the most mRNA destabilizing portion within CDK5R1 3'-UTR. This feature was maintained by a shorter region of 73 bp, characterized by two poly-U stretches. UV-CL experiments showed that this region interacts with protein factors. UV-CLIP assays and pull-down experiments followed by mass spectrometry analysis demonstrated that nELAV and hnRNPA2/B1 proteins bind to the same U-rich element. These RNA-binding proteins (RBPs) were shown to oppositely control CDK5R1 mRNA stability and p35 protein content at post-trascriptional level. While nELAV proteins have a positive regulatory effect, hnRNPA2/B1 has a negative action that is responsible for the mRNA destabilizing activity both of the C2.1 region and of the full-length 3'-UTR. In co-expression experiments of hnRNPA2/B1 and nELAV RBPs we observed an overall decrease of p35 content. We also demonstrated that hnRNPA2/B1 can downregulate nELAV protein content but not vice versa. This study, by providing new insights on the combined action of different regulatory factors, contributes to clarify the complex post-transcriptional control of CDK5R1 gene expression.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas ELAV/metabolismo , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas do Tecido Nervoso/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Western Blotting , Diferenciação Celular , Proteínas ELAV/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Imunoprecipitação , Luciferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
The human antiapoptotic bcl-2 gene has been discovered in t(14;18) B-cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl-2/IgH fusion gene. We were the first to disclose the post-transcriptional control of bcl-2 expression mediated by interactions of an adenine + uracil (AU)-rich element (ARE) in the 3'-UTR of bcl-2 mRNA with AU-binding proteins (AUBPs). Here, we identify and characterize zeta-crystallin as a new bcl-2 AUBP, whose silencing or overexpression has impact on bcl-2 mRNA stability. An increased Bcl-2 level observed in normal phytohemagglutinin (PHA)-activated T lymphocytes, acute lymphatic leukemia (ALL) T-cell lines, and T cells of patients with leukemia in comparison with normal non-PHA-activated T lymphocytes was concomitant with an increase in zeta-crystallin level. The specific association of zeta-crystallin with the bcl-2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl-2 mRNA and suggests a possible contribution of zeta-crystallin to bcl-2 overexpression occurring in this leukemia.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , zeta-Cristalinas/metabolismo , Western Blotting , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Fito-Hemaglutininas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , zeta-Cristalinas/antagonistas & inibidores , zeta-Cristalinas/genéticaRESUMO
In the 3'-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/genética , Linhagem Celular , Células Clonais , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Inativação Gênica , Genes Reporter , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Imunoprecipitação , Luciferases/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Antígeno-1 Intracelular de Células T , TransfecçãoRESUMO
We have identified previously a destabilizing adenine- and uracil-rich element (ARE) in the 3'-UTR of bcl-2 mRNA that interacted with ARE-binding proteins to down-regulate bcl-2 gene expression in response to apoptotic stimuli. We have also described three contiguous 2'-O-methyl oligoribonucleotides (ORNs) in both sense and antisense orientation with respect to the bcl-2 ARE that are able to regulate the bcl-2 mRNA half-life and Bcl-2 protein level in two different cell lines. Here we show that treatment of neuronal cell line (SHSY-5Y) with antisense ORNs targeting the bcl-2 ARE (bcl-2 ARE asORNs) prevents bcl-2 down-regulation in response to apoptotic stimuli with glucose/growth factor starvation (Locke medium) or oxygen deprivation and enhances the apoptotic threshold as evaluated by time-lapse videomicroscopy, fluorescence-activated cell sorting analysis, and caspase-3 activation. Additional effects of bcl-2 ARE asORNs included inhibition of cell cycle entry and a marked increase of cellular neurite number and length, a hallmark of neuronal differentiation resulting from bcl-2 up-regulation. The ability of bcl-2 ARE asORNs to enhance the apoptotic threshold and to induce neuronal differentiation implies their potential application as a novel informational tool to protect cells from ischemic damage and to prevent neuronal degeneration.
Assuntos
Adenina/fisiologia , Apoptose/fisiologia , Diferenciação Celular/genética , Neurônios/citologia , Oligorribonucleotídeos/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/metabolismo , Uracila/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Marcação de Genes/métodos , Humanos , Neurônios/fisiologia , Oligorribonucleotídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
BACKGROUND: CDK5R1 plays a central role in neuronal migration and differentiation during central nervous system development. CDK5R1 has been implicated in neurodegenerative disorders and proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3'-untranslated region (3'-UTR) suggests a role in post-transcriptional regulation of CDK5R1 expression. RESULTS: The bioinformatic study shows a high conservation degree in mammals and predicts several AU-Rich Elements (AREs). The insertion of CDK5R1 3'-UTR into luciferase 3'-UTR causes a decreased luciferase activity in four transfected cell lines. We identified 3'-UTR subregions which tend to reduce the reporter gene expression, sometimes in a cell line-dependent manner. In most cases the quantitative analysis of luciferase mRNA suggests that CDK5R1 3'-UTR affects mRNA stability. A region, leading to a very strong mRNA destabilization, showed a significantly low half-life, indicating an accelerated mRNA degradation. The 3' end of the transcript, containing a class I ARE, specifically displays a stabilizing effect in neuroblastoma cell lines. We also observed the interaction of the stabilizing neuronal RNA-binding proteins ELAV with the CDK5R1 transcript in SH-SY5Y cells and identified three 3'-UTR sub-regions showing affinity for ELAV proteins. CONCLUSION: Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3'-UTR and support the hypothesis that CDK5R1 gene expression is post-transcriptionally controlled in neurons by ELAV-mediated mechanisms. This is the first evidence of the involvement of 3'-UTR in the modulation of CDK5R1 expression. The fine tuning of CDK5R1 expression by 3'-UTR may have a role in central nervous system development and functioning, with potential implications in neurodegenerative and cognitive disorders.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Estabilidade de RNA , Regiões 3' não Traduzidas/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Estabilidade de RNA/genéticaRESUMO
The serine/threonine kinase mTOR, the major sensor of cell growth along the PI3K/Akt pathway, can be activated by agents acting on microtubules. Damaged microtubules induce phosphorylation of the Bcl-2 protein and lower the threshold of programmed cell death, both of which are inhibited by rapamycin. In HEK293 cells expressing Akt mutants, the level of Bcl-2 phosphorylation and the threshold of apoptosis induced by taxol or by nocodazole are significantly modified. In cells expressing dominant-negative Akt (DN-Akt), Bcl-2 phosphorylation and p70S6KThr421/Ser424 phosphorylation induced by taxol or nocodazole were significantly enhanced as compared to cells expressing constitutively active Akt (CA-Akt) and inhibited by rapamycin. Moreover, DN-Akt cells were more sensitive to antitubule agents than CA-Akt cells. In nocodazole-treated HEK293 cells sorted according to cell cycle, the p70S6KThr421/Ser424 phosphorylation was associated to the G2/M fraction. More relevant, nocodazole inhibited, in a dose-response manner, mTOR phosphorylation at Ser2448. This activity, potentiated in DN-Akt cells, was not detectable in CA-Akt cells. Our results suggest that death signals originating from damaged microtubules in G2/M can compete with G1 survival pathways at the level of mTOR. These findings have implications for cancer therapy and drug resistance.
Assuntos
Apoptose/fisiologia , Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fase G2/efeitos dos fármacos , Fase G2/fisiologia , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Mitose/fisiologia , Mutação , Nocodazol/farmacologia , Paclitaxel/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.
Assuntos
Linfoma de Burkitt/terapia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Catalítico/metabolismo , Apoptose , Sequência de Bases , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Regulação para Baixo , Genes Sintéticos , Humanos , Oligodesoxirribonucleotídeos Antissenso/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Catalítico/química , RNA Catalítico/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido RibonucleicoRESUMO
BACKGROUND: The role of the MYC oncogene in the apoptotic pathways is not fully understood. MYC has been reported to protect cells from apoptosis activation but also to sensitize cells to apoptotic stimuli. We have previously demonstrated that the down-regulation of Myc protein activates apoptosis in melanoma cells and increases the susceptibility of cells to various antitumoral treatments. Beyond the well-known role in the G1-->S transition, MYC is also involved in the G2-M cell cycle phases regulation. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have investigated how MYC could influence cell survival signalling during G2 and M phases. We used the microtubules damaging agent paclitaxel (PTX), to arrest the cells in the M phase, in a p53 mutated melanoma cell line with modulated Myc level and activity. An overexpression of Myc protein is able to increase endoreduplication favoring the survival of cells exposed to antimitotic poisoning. The PTX-induced endoreduplication is associated in Myc overexpressing cells with a reduced expression of MAD2, essential component of the molecular core of the spindle assembly checkpoint (SAC), indicating an impairment of this checkpoint. In addition, for the first time we have localized Myc protein at the spindle poles (centrosomes) during pro-metaphase in different cell lines. CONCLUSIONS: The presence of Myc at the poles during the prometaphase could be necessary for the Myc-mediated attenuation of the SAC and the subsequent induction of endoreduplication. In addition, our data strongly suggest that the use of taxane in antitumor therapeutic strategies should be rationally based on the molecular profile of the individual tumor by specifically analyzing Myc expression levels.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Replicação do DNA/fisiologia , DNA de Neoplasias , Melanoma/genética , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Western Blotting , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular , Regulação para Baixo , Fase G2/efeitos dos fármacos , Humanos , Melanoma/patologia , Mitose/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The in vitro efficacy of both EGFR inhibitor gefitinib and mTor inhibitor rapamycin, either administrated alone or in different combination schedules, was analysed in four pancreas cancer cell lines. Both drugs were found to induce cell growth inhibition, apoptosis as well as a slight but stable accumulation of cells in the G0/G1 phase. In all cell lines, neither gefitinib nor rapamycin affected EGFR and the expression of its downstream effectors. By contrast, gefitinib inhibited in a fast and completely way p-EGFR and partially p-Akt while a 3 days-rapamycin exposure resulted in the inhibition of the expression of both mTor and p70S6K. Moreover, after early stimulation, the mTor inhibitor produced a progressive, and almost complete inhibition of p-Akt. The analysis of combined gefitinib and rapamycin administration showed a clear schedule-dependent activity which turned out to be synergic only when gefitinib was given before rapamycin. This synergism seemed to depend on increase of both p-Akt and p70S6K inhibition, the greater the induction of apoptosis, the higher the decrease in cell cycle rate. Moreover, the antiangiogenic activity of the two drugs given in combination was demonstrated by a strong reduction of VEGF release which turned out to be more pronounced in the synergic schedule, and HIF-1alpha inhibition-independent. Our results suggest that the schedule of gefitinib followed by rapamycin, acting at different levels of the EGFR cellular pathway, could induce antitumor and antiangiogenic effects of clinical interest in the pancreas cancer model.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Sirolimo/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Neoplasias Pancreáticas/metabolismo , Proteínas Quinases/metabolismo , Serina-Treonina Quinases TOR , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs.
Assuntos
Oligorribonucleotídeos/farmacologia , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Estabilidade de RNA , RNA Mensageiro/análise , Regulação para Cima/genética , Adenina , Sequência de Bases , Proteínas Proto-Oncogênicas c-bcl-2/análise , UracilaRESUMO
BACKGROUND: In prostate cancer, mutations of the phosphatase PTEN can activate the kinase cascade PI3K/Akt/mTOR which induces drug resistance. METHODS: Chemosensitization by siRNA targeting Akt was studied in HEK293 cells forced to express CA-Akt or kinase-dead DN-Akt. To decrease drug resistance, Akt was silenced with siRNA in human prostate DU-145 cell line expressing the normal PTEN or in LNCaP and PC3 cell lines expressing mutated-PTEN. Taxol was used for the chemosensitization studies. RESULTS: Silencing Akt in the drug-resistant CA-Akt cells efficiently sensitized cells to antitubule agents, whereas silencing drug-responsive DN-Akt cells did not. Only minor effects were obtained in wild-type HEK293 cells. Potentiation by siRNA of taxol cytotoxicity was significantly greater in mutated-PTEN cells than in prostate cells expressing wild-type PTEN. The apoptotic program induced by taxol was preferentially potentiated by Akt siRNA in PTEN-mutated cell lines as regards the DU-145 cell line. CONCLUSIONS: Silencing Akt in PTEN-mutated prostate cancer cells enhances the antitumor effects of taxol. No siRNA chemosensitization was obtained in prostate cells with wild type PTEN.
Assuntos
Mutação/genética , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Masculino , Proteína Oncogênica v-akt/genética , Paclitaxel/farmacologia , Próstata/citologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Moduladores de Tubulina/farmacologiaRESUMO
RNA has become a promising target for pharmacological purposes. Most current strategies are directed toward down-regulating its functions. In this study, we provide evidence of the up-regulation of messenger RNA in a sequence-specific manner. The bcl2 (b)-ARE (adenine-uridine-rich element) in the 3'-untranslated region of the b-RNA that regulates the rate of RNA degradation has been targeted with three chemically modified oligoribonucleotides designed in the antisense orientation (asORNs). The three asORNs were studied by a cell-free degradation assay. All three slowed the rate of RNA decay in a dose-response fashion, they were specific to the b-ARE, and two of them were individually effective. asORNs were then transfected into the malignant cells in culture and b-RNA half-life was measured by real-time reverse transcriptase-polymerase chain reaction. We showed that by stabilizing b-RNA the three asORNs increased the expression of b-RNA and of the relevant protein in a dose-response fashion.
Assuntos
Adenina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Antissenso/farmacologia , Uridina/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
The mammalian target of rapamycin (mTOR) is a central regulator of ribosome biogenesis, protein synthesis, cell growth and neurite plasticity. The mTOR kinase controls the translation machinery, in response to amino acids and growth factors, via activation of p70 ribosomal S6 kinase (p70S6K) and inhibition of eIF-4E binding protein (4E-BP1). The mTOR protein belongs to the PI3K pathway activated by insulin, nutrients and growth factors. The PI3K pathway involves the Akt kinase, an upstream regulator of mTOR. Rapamycin is a potent immunosuppressant and investigational anticancer drug, which inhibits mTOR, blocking protein synthesis and arresting the cell cycle in G1 phase. A wide body of evidence supports the role of mTOR in cell signaling related to cell growth and proliferation. Nevertheless, our recent findings have revealed that mTOR may be also involved in a signaling pathway activated by microtubule-damaging drugs, including taxol and nocodazole. It is known that agents affecting the integrity of microtubules activate apoptotic program by inducing phosphorylation and inactivation of the antiapoptotic Bcl-2 protein in G2-M phase. We have some evidence that mTOR is involved in the enzymatic cascade that, starting from damaged microtubules, induces downstream phosphorylation of the Bcl-2 protein. We also found that the level of activity of Akt can regulate Bcl-2 phosphorylation, through the mTOR kinase. Since mTOR activation by survival signals occurs in G1 phase and damaged microtubules activate proapoptotic signals in G2-M phase, we suggest that mTOR might mediate these two different pathways in two different phases of the cell cycle.
Assuntos
Apoptose/fisiologia , Proteínas Quinases/metabolismo , Animais , Ciclo Celular/fisiologia , Morte Celular/fisiologia , Humanos , Proteínas Quinases/genética , Serina-Treonina Quinases TORRESUMO
Recent evidence suggests that gene expression may be regulated, at least in part, at post-transcriptional level by factors inducing the extremely rapid degradation of messenger RNAs. These factors include reactions between adenyl-uridyl-rich elements (AREs) of the relevant mRNA and either specific proteins that bind to these elements or exosomes. This review deals with examples of the proteins (AU-rich binding proteins, AUBPs) and exosomes, which have been shown to form complexes with AREs and bring about rapid degradation of the relevant mRNA, and with certain other factors, which protect the RNA from such degradation. The biochemical and physiological factors underlying the stability of messenger RNAs carrying the ARE motifs will be reviewed in the light of their emerging significance for cell physiology, human pathology, and molecular medicine. We also consider the possible application of the results of recent insights into the mechanisms to pharmacological interventions to prevent or cure disorders, especially developmental disorders, which the suppression of gene expression may bring about. Molecular targeting of specific steps in protein degradation by synthetic compounds has already been utilized for the development of pharmacological therapies.
Assuntos
Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Animais , Sistemas de Liberação de Medicamentos , Exorribonucleases/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Genéticos , RNA Mensageiro/química , Proteínas de Ligação a RNA/fisiologia , Sequências Reguladoras de Ácido NucleicoRESUMO
Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.
Assuntos
Genes bcl-2 , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Northern Blotting , Western Blotting , Caenorhabditis elegans , Linhagem Celular , Sequência Conservada , DNA Complementar/metabolismo , Bases de Dados como Assunto , Regulação para Baixo , Evolução Molecular , Regulação da Expressão Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , UrocordadosRESUMO
We previously identified a conserved A + U-rich element (ARE) in the 3'-untranslated region of bcl-2 mRNA. We have also recently demonstrated that the bcl-2 ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2 mRNA half-life reduction. Here we show that the AUBP AUF1 binds in vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1-257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to the bcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2-3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis.
Assuntos
Genes bcl-2 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Apoptose/fisiologia , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Regulação da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0 , Humanos , Células Jurkat , Cinética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We have shown previously that the decay of human bcl-2 mRNA is mediated by an adenine/uridine-rich element (ARE) located in the 3'-untranslated region. Here, we have utilized a non-radioactive cell-free mRNA decay system to investigate the biochemical and functional mechanisms regulating the ARE-dependent degradation of bcl-2 mRNA. Using RNA substrates, mutants, and competitors, we found that decay is specific and ARE-dependent, although maximized by the ARE-flanking regions. In unfractionated extracts from different cell types and in whole cells, the relative enzymatic activity was related to the amount of Bcl-2 protein expressed by the cells at steady state. The degradation activity was lost upon Bcl-2 depletion and was reconstituted by adding recombinant Bcl-2. Ineffective extracts from cells that constitutively do not express Bcl-2 acquire full degradation activity by adding recombinant Bcl-2 protein. We conclude that Bcl-2 is necessary to activate the degradation complex on the relevant RNA target.