RESUMO
Small RNAs modulate gene expression by forming a ribonucleoprotein complex with Argonaute proteins and directing them to specific complementary sites in target nucleic acids. However, the interactions required for the recruitment of the target nucleic acid to the ribonucleoprotein complex are poorly understood. In the present manuscript we have investigated this question by using let-7a, Argonaute2 and a fully complementary mRNA target. Importantly, we have found that recombinant Argonaute2 is sufficient to direct let-7a guided cleavage of mRNA. Thus this model system has allowed us to investigate the mechanistic basis of silencing in vitro and in vivo. Current models suggest that Argonaute proteins bind to both the 5' and 3' termini of the guide RNA. We have found that the termini of the let-7a microRNA are indeed critical, since circular let-7a does not support mRNA cleavage. However, the 5' end is the key determinant, since its deletion abrogates activity. Surprisingly, we have found that alteration of the 5' terminal uracil compromises mRNA cleavage. Importantly, we have found that substitution of this base has little effect upon the formation of the binary let-7a-Argonaute2 complex, but inhibits the formation of the ternary let-7a-Argonaute2-mRNA complex. Thus we conclude that the interaction of the 5' uracil base with Argonaute2 plays a critical and novel role in the recruitment of mRNA.
Assuntos
Regiões 5' não Traduzidas/genética , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , MicroRNAs/química , RNA Mensageiro/genética , Uracila/química , Proteínas Argonautas , Sequência Conservada/genética , Células HeLa , Humanos , MicroRNAs/metabolismo , Estrutura Terciária de Proteína/genética , RNA Mensageiro/química , Ribonucleoproteínas/biossíntese , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Uracila/fisiologia , Pequeno RNA não TraduzidoRESUMO
We examined associations between haplotypes of the serotonin 1B receptor gene and individual differences in anger and hostility. Data were analyzed from a study of 361 university students (47% male). Participants were genotyped at five polymorphisms in the HTR1B gene (rs11568817, rs130058, rs6296, rs6297, rs13212041), including promoter and 3'UTR polymorphisms with opposite functional effects on gene expression. Participants reported their emotional states across 30 consecutive days for up to 4 years. Haplotype pairs were constructed statistically and assigned to a level of HTR1B expression based on the presence of the functional polymorphisms. Six haplotypes accounted for >97% of chromosomes. Three low expression haplotypes contained the 3'UTR variant (rs13212041 A-allele) that enables a microRNA-mediated reduction in expression. One intermediate expression haplotype contained the 3'UTR A-allele paired with the high-activity promoter. Two high expression haplotypes contained the 3'UTR variant (rs13212041 G-allele) that attenuates microRNA-mediated reduction in expression. Men with low expression haplotypes reported greater anger and hostility than men with one or two high expression haplotypes. Diplotype classification accounted for 8.4% of the variance in men's anger and hostility, primarily due to the 3'UTR polymorphism (rs13212041), but with some contribution of the functional promoter combination (rs11568817, rs130058). Associations with anger and hostility were not found in women. These findings extend our understanding of the genetic basis of anger and hostility by showing that newly characterized HTR1B haplotypes, particularly those with rs13212041, which modulates microRNA-mediated regulation of HTR1B expression, may have important implications for aggression-related phenotypes among young men.
Assuntos
Ira , Hostilidade , Polimorfismo Genético , Receptor 5-HT1B de Serotonina/genética , Regiões 3' não Traduzidas , Adulto , Sequência de Bases , Primers do DNA , Genótipo , Haplótipos , Humanos , Masculino , Regiões Promotoras GenéticasRESUMO
Micro-RNAs are small noncoding RNAs, which diminish the stability and/or translation of mRNAs. This study examined whether miR-206, previously shown to be elevated in estrogen receptor (ER)alpha-negative breast cancer, regulates the expression of ERalpha. Two putative miR-206 sites, (hERalpha1 and hERalpha2), were found in silico within the 3'-untranslated region of human ERalpha mRNA. Transfection of MCF-7 cells with pre-miR-206 or 2'-O-methyl antagomiR-206 specifically decreased or increased, respectively, ERalpha mRNA levels. Overexpression of pre-miR-206 reduced ERalpha and beta-actin protein levels, with no effect on ERbeta, E-cadherin, or glyceraldehyde-3-phosphate dehydrogenase. Reporter constructs containing the hERalpha1 or hERalpha2 binding sites inserted into the 3'-untranslated region of the luciferase mRNA conferred a 1.6- and 2.2-fold repression of luciferase activity, respectively, in HeLa cells. Both miR-206 sites responded accordingly to exogenous hsa-pre-miR-206 and 2'-O-methyl antagomiR-206, and both sites were rendered inactive by mutations that disrupted hybridization to the 5'-seed of miR-206. A C-->T single nucleotide polymorphism in the hERalpha1 site increased repression of luciferase activity to approximately 3.3-fold in HeLa cells. MiR-206 levels were higher in ERalpha-negative MB-MDA-231 cells than ERalpha-positive MCF-7 cells, but only the ERalpha1 site mediated significantly more repression in reporter constructs. MiR-206 expression was strongly inhibited by ERalpha agonists, but not by an ERbeta agonist or progesterone, indicating a mutually inhibitory feedback loop. These findings provide the first evidence for the posttranscriptional regulation of ERalpha by a micro-RNA in the context of breast cancer.
Assuntos
Receptor alfa de Estrogênio/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Transcrição Gênica , Regiões 3' não Traduzidas , Sequência de Bases , Neoplasias da Mama , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , MicroRNAs/química , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Conformação de Ácido Nucleico , Biossíntese de ProteínasRESUMO
Angiogenesis, or new blood vessel formation, is critical for the growth and spread of tumors. Multiple phases of this process, namely, migration, proliferation, morphogenesis, and vascular stabilization, are needed for optimal tumor growth beyond a diffusion-limited size. The sphingosine 1-phosphate (S1P) receptor-1 (S1P(1)) is required for stabilization of nascent blood vessels during embryonic development. Here we show that S1P(1) expression is strongly induced in tumor vessels. We developed a multiplex RNA interference technique to downregulate S1P(1) in mice. The small interfering RNA (siRNA) for S1P(1) specifically silenced the cognate transcript in endothelial cells and inhibited endothelial cell migration in vitro and the growth of neovessels into subcutaneous implants of Matrigel in vivo. Local injection of S1P(1) siRNA, but not a negative control siRNA, into established tumors inhibited the expression of S1P(1) polypeptide on neovessels while concomitantly suppressing vascular stabilization and angiogenesis, which resulted in dramatic suppression of tumor growth in vivo. These data suggest that S1P(1) is a critical component of the tumor angiogenic response and argue for the utility of siRNA technology in antiangiogenic therapeutics.
Assuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Colágeno/metabolismo , Combinação de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Regulação Neoplásica da Expressão Gênica , Laminina/metabolismo , Camundongos , Transplante de Neoplasias , Neoplasias/metabolismo , Proteoglicanas/metabolismo , RNA Interferente Pequeno/genética , Receptores de Lisoesfingolipídeo/genética , Transplante HeterólogoRESUMO
HuR is a ubiquitously expressed mRNA-binding protein. Intracellular localization of HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm. In the cytoplasm it can stabilize certain transcripts. Because nucleocytoplasmic translocation of HuR is necessary for its activity, it was hypothesized that cytoplasmic HuR expression in cancer cells could be a prognostic marker. To test the significance of HuR in carcinogenesis of the breast, we have investigated HuR expression in a mouse mammary gland tumor model and from 133 invasive ductal breast carcinoma specimens. HuR expression was elevated in the cyclooxygenase-2 transgene-induced mouse mammary tumors, and its expression was predominantly cytoplasmic in the tumor cells. In the human carcinoma samples, high cytoplasmic immunoreactivity for HuR was found in 29% (38 of 133) of the cases. Cytoplasmic HuR expression associated with high grade (P = 0.0050) and tumor size over 2 cm (P = 0.0082). Five-year distant disease-free survival rate was 42% [95% confidence interval (95% CI), 26-58] in cytoplasm-high category and 84% (95% CI, 76-91) in cytoplasm-negative or -low category (P < 0.0001), and high cytoplasmic expression of HuR was an independent prognostic factor in a Cox multivariate model (relative risk 2.07; 95% CI, 1.05-4.07). Moreover, high cytoplasmic HuR immunopositivity was significantly associated with poor outcome in the subgroup of node-negative breast cancer in a univariate analysis (P < 0.0007). Our results show that high cytoplasmic HuR expression is associated with a poor histologic differentiation, large tumor size, and poor survival in ductal breast carcinoma. Thus, HuR is the first mRNA stability protein of which expression associates with poor outcome in breast cancer.
Assuntos
Antígenos de Superfície/biossíntese , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas de Ligação a RNA/biossíntese , Animais , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Citoplasma/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Análise Multivariada , PrognósticoRESUMO
Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3'-untranslated region (3'-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3'-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3'-UTR, which prolongs the mRNA lifetime and increases protein level.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Serpinas/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Encéfalo/metabolismo , Proteínas ELAV , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hibridização In Situ , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Células PC12 , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serpinas/genética , Transcrição Gênica , NeuroserpinaRESUMO
Cyclooxygenase-2 (COX-2) expression has been shown to associate with poor prognosis in ovarian cancer, and an mRNA stability protein HuR has been shown to enhance expression of COX-2 in tissue culture conditions. We found cytoplasmic immunoreactivity for HuR protein in 52% (233 of 445) of serous-type ovarian carcinoma specimens, and it associated with high COX-2 expression (P = 0.0045) and with clinicopathological variables, including poor prognosis (P < 0.0001) and high grade (P < 0.0001). In ovarian cancer cells in vitro, a small interfering RNA against HuR and leptomycin B, an inhibitor of nucleocytoplasmic translocation of HuR, inhibited COX-2 expression. Our results show that cytoplasmic HuR expression associates with poor outcome in ovarian cancer, and one plausible explanation for this finding may be related to the ability of HuR to induce COX-2 expression.
Assuntos
Antígenos de Superfície , Cistadenocarcinoma Seroso/metabolismo , Isoenzimas/biossíntese , Neoplasias Ovarianas/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteínas de Ligação a RNA/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ciclo-Oxigenase 2 , Cistadenocarcinoma Seroso/enzimologia , Citoplasma/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ácidos Graxos Insaturados/farmacologia , Feminino , Humanos , Isoenzimas/antagonistas & inibidores , Proteínas de Membrana , Pessoa de Meia-Idade , Análise Multivariada , Neoplasias Ovarianas/enzimologia , Prognóstico , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Análise de SobrevidaRESUMO
Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of monoclonal B lymphocytes in the hematopoietic organs. Rarely, CLL cells accumulate in a single atypical site. The mechanism underlying this unusual distribution of CLL cells has not been studied previously. We obtained peripheral blood from five patients having early stage CLL with heavy prostate infiltration. These patients' circulating CLL cells bound strongly in vitro to cultured prostate cell lines PC3, LNCaP, and DU145 and to short-term cultures of fresh prostate cells but not to colon, breast, or bladder cells. CLL cells from patients without prostate infiltration did not bind in vitro to any cell line. Peripheral blood CLL cells from one patient with CLL with heavy prostate infiltration were fused with a mouse-human heteromyeloma line to make hybridomas expressing the same monoclonal IgM as the patient's CLL cells. The hybridoma cells bound specifically to prostate cells. IgM secreted by the hybridoma blocked binding of the patient's CLL cells to prostate cells. Flow cytometry and immunohistochemistry demonstrated that the secreted IgM bound specifically to prostate cells. These results indicate that CLL with atypical prostate infiltration can be mediated by specific surface-bound IgM against an antigen expressed specifically by prostate cells and suggest that a similar mechanism might also apply to cases of CLL with atypical infiltration into other organs.
Assuntos
Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Infiltração Leucêmica/patologia , Próstata/patologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Citometria de Fluxo , Humanos , Hibridomas , Imunoglobulina M/imunologia , Imuno-Histoquímica , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Infiltração Leucêmica/imunologia , Infiltração Leucêmica/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Camundongos , Próstata/imunologia , Próstata/metabolismo , Células Tumorais CultivadasRESUMO
The RNA-binding protein HuR (also known as ELAV1) binds to the 3'-untranslated region of mRNAs and regulates transcript stability and translation. However, the in vivo functions of HuR are not well understood. Here, we report that murine HuR is essential for life; postnatal global deletion of Elavl1 induced atrophy of hematopoietic organs, extensive loss of intestinal villi, obstructive enterocolitis, and lethality within 10 days. Upon Elavl1 deletion, progenitor cells in the BM, thymus, and intestine underwent apoptosis, whereas quiescent stem cells and differentiated cells were unaffected. The survival defect of hematopoietic progenitor cells was cell intrinsic, as transplant of Elavl1-/- BM led to compromised hematopoietic reconstitution but did not cause lethality. Expression of p53 and its downstream effectors critical for cell death were induced in progenitor cells as HuR levels declined. In mouse embryonic fibroblasts, HuR bound to and stabilized the mRNA for Mdm2, a critical negative regulator of p53. Furthermore, cell survival was restored by expression of Mdm2 in Elavl1-/- cells, suggesting that HuR keeps p53 levels in check in progenitor cells and thereby promotes cell survival. This regulation of cell stress response by HuR in progenitor cells, which we believe to be novel, could potentially be exploited in cytotoxic anticancer therapies as well as stem cell transplant therapy.
Assuntos
Antígenos de Superfície/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Antígenos de Superfície/genética , Apoptose , Diferenciação Celular , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Feminino , Trato Gastrointestinal/citologia , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Deleção de Genes , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Tolerância a Radiação , Tamoxifeno/toxicidade , Quimeras de Transplante , Proteína Supressora de Tumor p53/metabolismoRESUMO
MicroRNAs are short, single-stranded RNAs that arise from a transient precursor duplex. We have identified a novel activity in HeLa cell extracts that can unwind the let-7 microRNA duplex. Using partially purified material, we have shown that microRNA helicase activity requires ATP and has a native molecular mass of approximately 68 kDa. Affinity purification of the unwinding activity revealed co-purification of P68 RNA helicase. Importantly, recombinant P68 RNA helicase was sufficient to unwind the let-7 duplex. Moreover, like its native homolog, P68 RNA helicase did not unwind an analogous small interfering RNA duplex. We further showed that knockdown of P68 inhibited let-7 microRNA function. From our data, we conclude that P68 RNA helicase is an essential component of the let-7 microRNA pathway, and in conjunction with other factors, it may play a role in the loading of let-7 microRNA into the silencing complex.
Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Trifosfato de Adenosina/metabolismo , Sequência de Bases , RNA Helicases DEAD-box/isolamento & purificação , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain- and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNA-mediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.
Assuntos
Ciclo-Oxigenase 2/genética , Implantação do Embrião/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Útero/metabolismo , Animais , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos , GravidezRESUMO
The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.
Assuntos
Antígenos de Superfície/metabolismo , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Sequência de Bases , Senescência Celular/efeitos dos fármacos , Quinase do Ponto de Checagem 2 , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Mutação Puntual/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Sirtuína 1 , Especificidade por Substrato/efeitos dos fármacosRESUMO
UNLABELLED: Cyclooxygenase-2 (COX-2) expression has been found to associate with poor prognosis in several types of carcinomas. HuR is an mRNA stability protein and it regulates the expression of COX-2. OBJECTIVES AND METHODS: We analyzed the expression of COX-2 and HuR in 64 mucinous ovarian carcinoma specimens by immunohistochemistry. RESULTS: In mucinous tumors, high COX-2 protein expression was found in epithelial cancer cells in 39% (22/56) and in stromal cells in 24% (13/55) of the specimens. The expression of COX-2 in cancer cells correlated with high grade (P = 0.0285), but stromal COX-2 expression had no correlation with any clinical parameter tested. Cytoplasmic HuR protein expression was observed in cancer cells in 47% (27/57) and in stromal cells in 7% (4/56) of the mucinous tumors, and it correlated with COX-2 expression in the cancer cells (P = 0.0162) but not in the stroma. CONCLUSION: Our results support the hypothesis that cytoplasmic HuR is connected to COX-2 expression in ovarian carcinoma, but that its role is restricted to the transformed epithelial cancer cells.
Assuntos
Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patologia , Antígenos de Superfície/biossíntese , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteínas de Ligação a RNA/biossíntese , Adenocarcinoma Mucinoso/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ciclo-Oxigenase 2 , Citoplasma/enzimologia , Citoplasma/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Proteínas de Membrana , Pessoa de Meia-Idade , Neoplasias Ovarianas/enzimologia , Células Estromais/enzimologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.
Assuntos
Antígenos de Superfície , Aorta/enzimologia , Processamento Pós-Transcricional do RNA , Receptores Citoplasmáticos e Nucleares/biossíntese , Regiões 3' não Traduzidas , Animais , Aorta/metabolismo , Aorta/patologia , Sequência de Bases , Northern Blotting , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Dactinomicina/farmacologia , Regulação para Baixo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ativadores de Enzimas/farmacologia , Guanilato Ciclase , Indazóis/farmacologia , Masculino , Dados de Sequência Molecular , Músculo Liso/citologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oxidiazóis/farmacologia , Oxazinas/farmacologia , Poli A/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Guanilil Ciclase Solúvel , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
Many genes governed by thyroid hormone (T3) lack binding sites for its receptor (TR) and are thought to be post-transcriptionally regulated by T3. Here we demonstrate that the HuD gene, which encodes a neurone-specific protein that binds to mRNA and modulates its stability, is regulated by T3. HuD RNA and protein expression were strongly up-regulated in specific areas of the hypothyroid rat brain, and reduced by T3 in rat PC12 and mouse N2a cells containing appropriate TR levels. Furthermore, T3 inhibited the transcription of HuD in run-on assays. Finally, HuD protein bound with high affinity to two sequences in acetylcholinesterase mRNA, and ectopic HuD expression increased its abundance in N2a cells. This is the first report of a gene encoding an mRNA stability regulator that is under T3 control. The results suggest that HuD may mediate some T3 effects by altering the half-life of mRNAs for acetylcholinesterase and other genes.
Assuntos
Proteínas do Tecido Nervoso/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transcrição Gênica/fisiologia , Tri-Iodotironina/fisiologia , Acetilcolinesterase/genética , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Encéfalo/metabolismo , Linhagem Celular , Proteínas ELAV , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipotireoidismo/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Células PC12 , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Receptores dos Hormônios Tireóideos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Tri-Iodotironina/farmacologiaRESUMO
The neuron-specific ELAV/Hu family member, HuD, interacts with and stabilizes GAP-43 mRNA in developing neurons, and leads to increased levels of GAP-43 protein. As GAP-43 protein is enriched in growth cones, it is of interest to determine if HuD and GAP-43 mRNA are associated in developing growth cones. HuD granules in growth cones are found in the central domain that is rich in microtubules and ribosomes, in the peripheral domain with its actin network, and in filopodia. This distribution of HuD granules in growth cones is dependent on actin filaments but not on microtubules. GAP-43 mRNA is localized in granules found in both the central and peripheral domains, but not in filopodia. Ribosomes were extensively colocalized with HuD and GAP-43 mRNA granules in the central domain, consistent with a role in the control of GAP-43 mRNA stability in the growth cone. Together, these results demonstrate that many of the components necessary for GAP-43 mRNA translation/stabilization are present within growth cones.
Assuntos
Proteína GAP-43/fisiologia , Cones de Crescimento/fisiologia , Proteínas do Tecido Nervoso/fisiologia , RNA Mensageiro/fisiologia , Proteínas de Ligação a RNA/fisiologia , Ribossomos/fisiologia , Animais , Proteínas ELAV , Proteína GAP-43/biossíntese , Proteína GAP-43/genética , Cones de Crescimento/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Células PC12 , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Ratos , Ribossomos/efeitos dos fármacos , Ribossomos/genéticaRESUMO
The cyclooxygenase-2 (COX-2) gene encodes the inducible prostaglandin synthase enzyme implicated in inflammation, cell growth, and tumorigenesis. Regulation of the COX-2 gene expression at the post-transcriptional level is poorly understood. For example, protein factors that regulate the post-transcriptional mRNA metabolism of COX-2 have not been fully characterized. In this study, we demonstrate that the RNA-binding protein HuR binds to COX-2 mRNA and regulates its expression. We show that there are three binding sites for HuR in the 3'-untranslated region of human COX-2. These sites are located at the following positions in the COX-2 3'-untranslated region: 39-84 nucleotides (nt), 1155-1187 nt, and 1244-1256 nt (hereinafter referred to as Sites I, II and III, respectively). Although all three sites are present in the 4.6-kb COX-2 mRNA, only site I is present in the shorter 2.8-kb isoform. HuR in MDA-MB-231 cell extracts associated with COX-2 mRNA at the identified sites. Further, HuR location in the cytoplasm was induced by serum withdrawal, a stimulus known to induce COX-2 mRNA. Down-regulation of HuR by two independent methods, namely RNA interference as well as antisense RNA expression, significantly attenuated serum withdrawal-induced increase in COX-2 mRNA (both the 4.6- and 2.8-kb isoforms) and protein levels. These data suggest that HuR binding to COX-2 is critical for its post-transcriptional mRNA stabilization.
Assuntos
Antígenos de Superfície , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Proteínas de Ligação a RNA/genética , Sequência de Bases , Ciclo-Oxigenase 2 , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/biossíntese , Proteínas de Membrana , Dados de Sequência Molecular , Prostaglandina-Endoperóxido Sintases/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21(WAF1) is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21(WAF1) mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21(WAF1) 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21(WAF1) 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21(WAF1) 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21(WAF1) mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21(WAF1) protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21(WAF1) mRNA via different mechanisms. We conclude that EGF-induced p21(WAF1) protein expression is mediated largely by stabilization of p21(WAF1) mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21(WAF1) expression or growth inhibition in this system. CP1 is a novel p21(WAF1) mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21(WAF1) mRNA stability.
Assuntos
Regiões 3' não Traduzidas/genética , Neoplasias da Mama/genética , Ciclinas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Poli C/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Cinética , Luciferases/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The androgen receptor (AR) mediates androgen action and plays a central role in the proliferation of specific cancer cells. We demonstrated recently that AR mRNA stability is a major determinant of AR gene expression in prostate and breast cancer cells and that androgens differentially regulate AR mRNA decay dependent on cell type (Yeap, B. B., Kreuger, R. G., Leedman, P. J. (1999) Endocrinology 140, 3282-3291). Here, we have identified a highly conserved UC-rich region in the 3-untranslated region of AR mRNA that contains a 5'-C(U)(n)C motif and a 3'-CCCUCCC poly(C)-binding protein motif. In transfection studies with LNCaP human prostate cancer cells, the AR UC-rich region reduced expression of a luciferase reporter gene. The AR UC-rich region was a target for cytoplasmic and nuclear RNA-binding proteins from human prostate and breast cancer cells as well as human testicular and breast cancer tissue. One of these proteins is HuR, a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins involved in the stabilization of several mRNAs. Poly(C)-binding protein-1 and -2 (CP1 and CP2), previously implicated in the control of mRNA turnover and translation, also bound avidly to the UC-rich region. Mutational analysis of the UC-rich region identified specific binding motifs for both HuR and the CPs. HuR and CP1 bound simultaneously to the UC-rich RNA and in a cooperative manner. Immunoprecipitation studies confirmed that each of these proteins associated with AR mRNA in prostate cancer cells. In summary, we have identified and characterized a novel complex of AR mRNA-binding proteins that target the highly conserved UC-rich region. The binding of HuR, CP1, and CP2 to AR mRNA suggests a role for each of these proteins in the post-transcriptional regulation of AR expression in cancer cells.