A expressão de Smads e de membros da família AP1/ jun-fos podem refletir alterações da via de TGFb, uma via importante para o câncer epidermóide de cabeça e pescoço (HNSCC). Encontramos expressão aumentada dos mRNAs das Smads1-8 em HNSCC em comparação com tecido normal adjacente, por RPA. Além disso, as curvas de sobrevida de Kaplan Meier e a análise multivariada mostraram que a Smad6+ parece ser um fator determinante de bom prognóstico em HNSCC. Quanto a família AP-1, mensurado por Northern blot, somente Fra-1 mostrou-se aumentado no tumor e associado à presença de linfonodos comprometidos. Nossos dados sugerem que a positividade de Smad6 possa ser marcador de bom prognóstico em HNSCC ; Smad and AP1 messenger RNA expression may underlie disruptions affecting TGFb signaling in head and neck squamous cell carcinoma (HNSCC). Analysis of Smads1-8 mRNA expression by RPA has shown Smad expression is globally increased in tumor as compared to adjacent normal tissue. Kaplan Meier survival curves and multivariate analysis revealed that Smad6 positivity in tumor was an independent good prognostic factor in HNSCC. In relation to AP-1, as measured by Northern blot, only Fra-1 was overexpressed in tumor and directly related to the presence of lymph node involvement. Our data suggest that Smad6 may be a marker of good prognosis in HNSCC
Tamoxifen was proven to reduce the incidence of breast cancer by 49% in women at increased risk of the disease in the Breast Cancer Prevention Trial. In order to identify potential candidates to explain the preventive effect induced by tamoxifen on breast cancer, normal breast tissue obtained from 42 fibroadenoma patients, randomly assigned to receive placebo or tamoxifen, was analyzed by the reverse Northern blot and RT-PCR techniques. The cDNA fragments used on Northern blot membranes were generated by the Human Cancer Genome Project funded by the Ludwig Institute for Cancer Research and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil). Total RNA was obtained from normal breast tissue from patients with clinical, cytological and ultrasound diagnosis of fibroadenoma. After a 50-day treatment with tamoxifen (10 or 20 mg/day) or placebo, normal breast tissue adjacent to the tumor was collected during lumpectomy with local anesthesia. One differentially expressed gene, Calcium/calmodulin-dependent protein kinase II (CaMKII), was found to be down-regulated during TAM treatment. CaMKII is an ubiquitous serine/threonine protein kinase that has been implicated in the diverse effects of hormones utilizing Ca2+ as a second messenger as well as in c-fos activation. These results indicate that the down-regulation of CaMKII induced by TAM might represent alternative or additional mechanisms of the action of this drug on cell cycle control and response to hormones in normal human breast tissue.
Human hemoglobin messenger RNA was isolated by sucrose gradient centrifugation from reticulocytes of patients having various hemolytic anemias. Using a messenger RNA-dependent cell-free system derived entirely from rabbit reticulocytes, the human hemoglobin messenger RNA has been translated and the products analyzed by carboxymethylcellulose column chromatography. Normal messenger RNA directs synthesis of normal human α- and β-globin chains in nearly equal amounts. Sickle cell anemia messenger RNA directs the synthesis of normal α- and sickle β-chains, β-thalassemia messenger RNA directs the synthesis of normal α- and β-chains, but the amount of β-globin synthesized is markedly reduced. Thus the inability of the thalassemia reticulocyte to produce β-globin is clearly attributable to the β-globin messenger RNA.
Functional messenger RNA for human hemoglobin synthesis was prepared from reticulocyte lysates of patients with homozygous beta thalassemia and sickle cell anemia. The messenger RNA stimulated the synthesis of human globin chains by a cell-free system derived from Krebs mouse ascites cells. In the presence of beta thalassemia messenger RNA, the system synthesized much less beta chain than alpha chain whereas in the presence of sickle cell anemia messenger RNA, nearly equal amounts of alpha and beta chains were synthesized. The beta/alpha synthetic ratios obtained in the cell-free system were similar to those obtained by incubating intact beta thalassemia and sickle cell anemia reticulocytes in the presence of radioactive leucine. The experiments provide direct evidence of a defect in messenger RNA for beta chains as a cause for the decreased synthesis of beta chains observed in beta thalassemia.
The appearance of messenger RNA for myosin heavy chains in chick-embryo myogenic cell cultures was investigated. Total polyribosomes were isolated from cultures at various times of development and were purified in sucrose step gradients. These polysomes were either extracted with phenol or were treated with puromycin. The ribonucleoprotein particles and ribosomal subunits released by puromycin were fractionated on sucrose gradients. RNA from polysomes or from puromycin-dissociated subunits was fractionated on oligo(dT)-cellulose columns, and the bound and unbound RNA was assayed for activity of myosin heavy chain messenger RNA in a rabbit reticulocyte cell-free system. RNA stimulating myosin heavy-chain synthesis was found predominantly in the unbound fractions of the oligo(dT)-cellulose columns. After puromycin treatment of polysomes, the myosin heavy chain messenger RNA, which sediments at 18-26 S, was associated with a ribonucleoprotein particle sedimenting between 30 and 40 S. Myosin heavy chain messenger RNA was obtained from cultures containing well-developed myotubes and from cultures undergoing myogenic cell fusion. This messenger RNA was not detectable in early, unfused cultures, or in later cultures in which myogenic cell fusion had been prevented by treatment with ethyleneglycol bis(β-aminoethyl ether)-N...
The association between certain cellular RNAs and purified RNA tumor viruses prompted us to examine the possibility that specific host messenger RNAs might also be incorporated into RNA tumor viruses. Using a mouse cell line infected with Friend leukemia virus, T-3-Cl-2, which can be induced to accumulate mouse-globin messenger RNA, we show that mouse-globin messenger RNA sequences are present in viral particles purified from the culture medium of globin-producing cells. These globin messenger RNA sequences are absent from viral particles derived from T-3-Cl-2 cells that are not producing globin messenger RNA. Virus-associated globin messenger RNA sequences sediment in association with the 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin messenger RNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin messenger RNA does not become associated with the 60S viral RNA complex; and that globin messenger RNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin messenger RNA.
A mammalian cell-free transcriptional system was developed in which mammalian RNA polymerase synthesizes globin messenger RNA sequences from bone-marrow chromatin. The messenger RNA sequences are detected by measurement of the ability of the transcribed RNA to hybridize with globin complementary DNA. The globin complementary DNA is synthesized by the enzyme from avian myeloblastosis virus, RNA-directed DNA polymerase, with purified globin messenger RNA as template. The specificity of the globin complementary DNA in annealing reactions was verified by preparing DNA complementary to liver messenger RNA and showing that the globin and liver complementary DNAs are specific for their own messenger RNAs. Both DNA-dependent RNA polymerase II from sheep liver and RNA polymerase from Escherichia coli can transcribe globin messenger RNA sequences from rabbit bone-marrow chromatin; however, the mammalian enzyme appears to be more specific in that globin gene sequences represent a higher proportion of the RNA synthesized. Neither polymerase can transcribe globin messenger RNA sequences from rabbit-liver chromatin. This cell-free assay system should be useful in searching for mammalian transcriptional regulatory factors.
The rate of synthesis of phosphoenolpyruvate carboxykinase (EC 22.214.171.124) in rat liver decreased with a half-life of 30 min after fasted rats were refed either a chow diet or glucose. A requirement for both glucose and insulin to bring about this rapid deinduction was shown, as well as the ability of dibutyryl adenosine 3′:5′-cyclic monophosphate to block the decrease in enzyme synthesis. Estimates of the stability of messenger RNA of phosphoenolpyruvate carboxykinase were made by using the inhibitors actinomycin D and cordycepin to block further messenger RNA synthesis, and then measuring the decrease in specific enzyme synthesis. It is suggested that the use of actinomycin D yields an overestimation of the template stability. The results with cordycepin imply that the enzyme messenger RNA has a short half-life of approximately 1 hr. Thus, it is possible that deinduction may proceed by way of a decrease in messenger RNA production, leading to a rapid fall in the synthesis of phosphoenolpyruvate carboxykinase.
The synthesis of poly(A)-containing cytoplasmic RNA was examined in primary myoblast cultures prepared from skeletal muscle of fetal calves. After a period of cell division, these cells undergo fusion, with concomitant appearance of acetylcholine receptor and subsequent myosin synthesis. In the dividing myoblast there is a high level of messenger RNA synthesis, including a 26S RNA, the size of a putative messenger for the large subunit of myosin. In the transition period prior to fusion, there are quantitative changes in RNA synthesis. At this time, there is a pronounced production of 26S RNA, which diminishes during fusion. The possibility that 26S RNA is accumulated in the dividing myoblast was investigated by chase experiments. At fusion, there is a marked increase in the half-lives of a number of messenger RNA species, including 26 S, which increases from about 10 hr in the dividing cell to a value of more than 50 hr. The identity of the more rapidly turning over 26 S in the myoblasts, compared to that of the 26 S at fusion, was examined in terms of polysomal distribution, migration on gels, and hybridization with complementary DNA for the myosin message. The results of these analyses suggest that the 26S species are identical. Thus...
It is shown that initiation of translation involves several steps. (i) Binding of fMet-tRNAfMet to the bacterial 30S ribosomal subunit in the absence of messenger RNA, yielding a 34S complex. This binding is rapid and dependent on initiation factor 2 but not on initiation factor 3. (ii) Binding of messenger RNA to the 34S complex. This binding is slower and depends on initiation factor 3. If R17 RNA is used as messenger, the resulting complex sediments at 46 S. (iii) Joining of a 50S subunit to yield a complete initiation complex. Binding of fMet-tRNAfMet not only precedes, but is necessary for, correct binding of messenger RNA to ribosomes. Thus, initiator tRNA may play an active role in the selection of initiation sites in messenger RNA.
Translation of messenger RNA isolated from poly(rI)-poly(rC)-induced human fibroblasts in cell-free ribosomal systems and in Xenopus oocytes resulted in the production of biologically active proteins that had the properties of human fibroblast interferon. The translation in the oocytes was much more efficient, giving approximately 500 times higher titers of interferon activity than the cell-free systems. A control messenger RNA isolated from noninduced human fibroblasts, did not code for interferon synthesis in these systems. Both messenger RNA preparations stimulated [3H]amino-acid incorporation into trichloroacetic acid-insoluble material. The radioactive products and their immunoprecipitates were electrophoresed on polyacrylamide gels under denaturing conditions. The products resulting from the translation of the control (uninduced) messenger RNA in oocytes contained a major protein of approximately 45,000 molecular weight. The messenger RNA isolated from poly(rI)-poly(rC)-induced cells stimulated the synthesis of an additional 25,000 molecular weight protein that electrophoresed in the same position as human fibroblast interferon. These results suggest that human fibroblast interferon was synthesized by the translation of its messenger RNA in Xenopus oocytes and in cell-free ribosomal systems.
Kinetics of polysome formation, translational capacity, and coding properties of polysome-associated messenger RNA were investigated in potato tuber tissue discs during aging. Polysome content rapidly increased immediately after slicing from 14% of total ribosomes in freshly sliced discs to 55% within 12 hours of aging. The amount of polysomal RNA also increased 5-fold during this period. Translational capacity of polysome-associated messenger RNA increased in parallel with the increase in content of polysomal RNA of the tissue discs when measured in a wheat germ cell-free system. Analysis of the in vitro translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the majority of polypeptides coded by the messenger RNA did not vary greatly during the period of rapid polysome formation. Three types of messenger RNA were found to change in amount during that period: those which appeared only after aging, those which disappeared during aging, and those which disappeared early but reappeared later in the aging period.
Cell monolayers wre treated with interferon, and then were labeled with RNA precursors. Messenger ribonucleoprotein and transfer RNA were liberated from isolated polysomes, and analyzed by electrophoresis on polyacrylamide gels. The messenger ribonucleoproteins and transfer RNA from interferon-treated cells migrated slightly slower than did the corresponding components from control cells. It was shown that the differences in migration speeds was attributable to the fact that the messenger RNAs and transfer RNA that were polysomebound (i.e., being translated) were slightly larger than control-cell RNAs. This phenomenon was seen in mouse L cells, primary mouse-embryo cells, and rabbit-kidney cells. Ribosomal 28S and 18S RNA was not altered, nor was nuclear RNA. Several types of evidence indicate that the biochemical changes are attributable to the interferon in the preparations, and not to impurities. These data may bear on the mechanism by which interferon-treated cells recognize and translate viral RNA poorly. It is tentatively suggested that interferon treatment of cells leads to a change in cellular messenger RNA such that the cell can distinguish it from the subsequently infecting viral RNA.
Messenger RNA from rat liver was partially purified by chromatography on cellulose on the basis of its poly(A) content. Microgram amounts of this RNA stimulate protein synthesis manyfold in a heterologous cell-free system, derived from Krebs ascites cells supplemented with reticulocyte initiation factors. The messenger RNA directs the initiation, synthesis, and release of a product that was identified as complete subunits of hepatic tryptophan oxygenase (EC 126.96.36.199) by immunoprecipitation with monovalent antibodies prepared against homogeneous tryptophan oxygenase and subsequent sodium dodecyl sulfate-polyacrylamide electrophoresis of the solubilized immunoprecipitate. This may represent the first complete translation in a heterologous system of a mammalian messenger RNA coding for an enzyme protein. Analysis of the messenger RNA content of the liver after glucocorticoid administration demonstrates that the hormonally enhanced rate of synthesis of tryptophan oxygenase is accompanied by an increased quantity of its corresponding messenger RNA.
The messenger RNA for ovalbumin, the major secretory protein of the chick oviduct, appears not to be made as a high-molecular-weight precursor when artifacts due to aggregation are eliminated. No ovalbumin messenger RNA sequences that will hybridize to complementary DNA made against ovalbumin mRNA are found in concentrated samples of hen oviduct RNA larger than 28 S. The sensitivity of the hybridization assay is sufficient to detect less than one molecule of ovalbumin mRNA precursor per tubular gland cell. Newly synthesized ovalbumin messenger RNA isolated from immature chicks stimulated briefly by estrogen is the same size as that found in hen polyribosomes. We conclude that ovalbumin messenger RNA does not undergo any significant change in molecular weight from its initial transcription to its incorporation into polyribosomes.
1. Histidine decarboxylase in the enterochromaffin-like cells of the gastric corpus mucosa converts histidine to histamine which in turn stimulates gastric acid secretion. The control of histidine decarboxylase activity is poorly understood. We have examined how fasting and refeeding influence the abundance of the messenger RNA encoding histidine decarboxylase in the gastric corpus of the rat. 2. The polymerase chain reaction was used to generate a probe for detection of histidine decarboxylase messenger RNA in Northern and slot blots of total RNA from the gastric corpus of rats fasted for up to 48 h, or fasted and then refed. A gastrin monoclonal antibody was used to neutralize the action of endogenous gastrin. 3. Fasting progressively reduced histidine decarboxylase messenger RNA abundance by 3- to 4-fold after 48 h. Refeeding induced a rapid increase in histidine decarboxylase messenger RNA abundance which was detectable after 30 min. 4. There was a significant correlation between histidine decarboxylase messenger RNA abundance and plasma gastrin. Administration of gastrin antibody inhibited the increase in histidine decarboxylase activity after 6 h refeeding, but not after refeeding for 30 min. 5. The results suggest that histamine-mediated changes in postprandial acid secretion depend on control of histidine decarboxylase mRNA levels...
Fe2+ is now shown to weaken binding between ferritin and mitochondrial aconitase messenger RNA noncoding regulatory structures ((iron-responsive element) (IRE)-RNAs) and the regulatory proteins (IRPs), which adds a direct role of iron to regulation that can complement the well known regulatory protein modification and degradative pathways related to iron-induced mRNA translation. We observe that the Kd value increases 17-fold in 5′-untranslated region IRE-RNA·repressor complexes; Fe2+, is studied in the absence of O2. Other metal ions, Mn2+ and Mg2+ have similar effects to Fe2+ but the required Mg2+ concentration is 100 times greater than for Fe2+ or Mn2+. Metal ions also weaken ethidium bromide binding to IRE-RNA with no effect on IRP fluorescence, using Mn2+ as an O2-resistant surrogate for Fe2+, indicating that metal ions bound IRE-RNA but not IRP. Fe2+ decreases IRP repressor complex stability of ferritin IRE-RNA 5–10 times compared with 2–5 times for mitochondrial aconitase IRE-RNA, over the same concentration range, suggesting that differences among IRE-RNA structures contribute to the differences in the iron responses observed in vivo. The results show the IRE-RNA·repressor complex literally responds to Fe2+, selectively for each IRE-mRNA.
Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3' untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3' poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3' UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work...
Accumulation of ovalbumin messenger RNA in chick oviduct is absolutely dependent upon estrogen. After estrogen treatment, ovalbumin comprises 60-65% of the total oviduct protein. We used maximally stimulated animals to extract and partially purify the ovalbumin messenger RNA. The final product was enriched about 100-fold in activity with respect to this specific messenger RNA. This ovalbumin messenger RNA fraction was used to direct the synthesis of a complementary [3H]DNA in the presence of RNA-dependent DNA polymerase isolated from avian myeloblastosis virus. The complementary [3H]DNA (specific radioactivity, 8 × 107 cpm/μg) was a faithful transcript since about 90% would hybridize back to the original messenger RNA template. Ovalbumin complementary [3H]DNA was reannealed with an excess of chick-oviduct total DNA. The kinetics of this reaction indicate that only one copy of the ovalbumin gene exists in each haploid genome. These data suggest that estrogen may affect the oviduct genome to stimulate production of large numbers of ovalbumin messenger RNA molecules from a single copy of the ovalbumin gene.
A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in frame translation stop codon. NF1 editing occurs in normal tissue but was several-fold higher in tumors. In vitro editing and transfection assays demonstrated that apoB and NF1 RNA editing will take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing did not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit) suggesting that different trans-acting factors may be involved in the two editing processes.; Article may be found at: http://nar.oxfordjournals.org/cgi/content/abstract/24/3/478; This work was supported in part by grants from the NIH (CA55173) and from Cancer Action, Inc. (to G.R.S.) and by the NIH (DK43739) and The Council for Tobacco Research (to H.C.S.). M.S. is supported through an American Heart Association (NY state affiliate) fellowship. L.J.M. and A.J.C. are supported in part by a predoctoral award from the Interdepartmental Training Grant in Genetics and Regulation (GM07102).