RESUMEN
We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.
Asunto(s)
Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Glutatión Transferasa/genética , Células HeLa , Humanos , Poli A/biosíntesis , Polinucleotido Adenililtransferasa/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/aislamiento & purificación , Factores de Escisión y Poliadenilación de ARNmRESUMEN
The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.
Asunto(s)
Poli A/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Virus 40 de los Simios/genética , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Transcripción GenéticaRESUMEN
Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.
Asunto(s)
VIH-1/genética , Poli A/metabolismo , ARN Viral/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Viral/genéticaRESUMEN
The human hepatitis B viral (HBV) genome contains a conserved open reading frame known as the X-gene which is capable of encoding a polypeptide of 16.565 kDa. The corresponding protein has so far not been identified directly in HBV-infected cells, but in transient transfection assays the X-gene encodes a product that functions as a transcriptional transactivator. To characterize the subcellular distribution, stability and post-translational modifications of X-protein in human hepatoma HepG2 cells, we have established a vaccinia virus expression system. As the major X-gene product, a protein with an apparent molecular weight of 16 kDa, and reacting with an X-protein-specific antiserum, was expressed from recombinant vaccinia virus. In indirect immunofluorescence assay, X-protein appeared to be distributed throughout the cells, with a tendency to localize at the nuclear periphery and to accumulate in granules as its levels increased. By subcellular fractionation, we found about one-third of X-protein associated with the fraction defined as the nuclear framework. In pulse-chase experiments, X-protein decayed with a bimodal half-life of 15 min and 3 h. X-protein having a half-life of about 15 min was found associated with the Triton X-100 detergent-soluble fraction of HepG2 cells, while that associated with the insoluble fraction turned over more slowly. By metabolic labeling with [32P] orthophosphate, we show that X-protein is capable of being phosphorylated. Modification by phosphorylation could play an important role in the regulation of X-protein function.
Asunto(s)
Transactivadores/metabolismo , Virus Vaccinia/metabolismo , Secuencia de Aminoácidos , Codón , Genoma Viral , Humanos , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Fosforilación , Transactivadores/genética , Transfección , Células Tumorales Cultivadas/metabolismo , Virus Vaccinia/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
An accelerated rate of glucose transport and catabolism is a common characteristic of cellular transformation. We have previously found elevated expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human pancreatic and colonic adenocarcinomas (Schek et al.: Cancer Res 48:6354-6359, 1988). To investigate further the expression of this enzyme in the process of tumorigenesis, we examined GAPDH expression in a panel of oncogene-transformed fibroblasts. Significant elevations of GAPDH mRNA and glucose transporter protein mRNA levels were observed in ras- and mos-transformed NIH 3T3 cells, whereas little or no change was found in c-src-, v-src-, c-myc-, E1A-, v-fos-, and PKC-gamma-transfected cells. Furthermore, the level of GAPDH mRNA correlated with the transformed state in a series of ras-transformed and revertant cell lines. Immunoblot analysis confirmed that GAPDH polypeptide was significantly elevated in the cell lines with elevated mRNA levels. Cell cycle analysis data suggested that the effect on GAPDH expression correlated with oncogene expression rather than cell growth fraction. These results suggest that altered GAPDH gene expression occurs during some growth deregulated states, and this, along with increased glucose transporter (and possibly other glycolytic enzyme) expression, is likely to contribute to the increased metabolic capacity of cells in these states.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Glucólisis , Oncogenes , Animales , Northern Blotting , Ciclo Celular , Línea Celular Transformada , Sondas de ADN , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Proteínas de Transporte de Monosacáridos/biosíntesis , Proteínas de Transporte de Monosacáridos/genética , ARN Mensajero/biosíntesisRESUMEN
To identify and characterize genes, the products of which play a role in pancreatic adenocarcinoma, we constructed a complementary DNA (cDNA) library using mRNA from the pancreatic adenocarcinoma cell line HPAF, grown as a nude mouse tumor. Through differential screening, we identified a cDNA clone, pII5B, that is homologous to an mRNA expressed at significantly higher levels in HPAF cells than in normal human pancreas. The pII5B cDNA was homologous to the 3'-untranslated region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12)mRNA. Partial sequencing of several HPAF tumor GAPDH cDNA clones revealed no significant differences from previously published GAPDH cDNA sequences. Increased levels of GAPDH mRNA, relative to actin mRNA levels, were found in six pancreatic adenocarcinoma cell lines and two nude mouse tumors, when compared to normal pancreas. Enolase and glucose transporter mRNA levels were also increased in HPAF cells and nude mouse tumor, suggesting a general increase in expression of genes associated with glycolysis in pancreatic adenocarcinoma. Levels of GAPDH protein were elevated in nude mouse tumors and fresh human pancreatic adenocarcinomas compared to normal pancreas. High GAPDH levels may be characteristic of human adenocarcinomas, since colon adenocarcinomas also exhibited high levels of GAPDH compared to normal colon.
Asunto(s)
Adenocarcinoma/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Neoplasias Pancreáticas/enzimología , Adenocarcinoma/genética , Animales , Secuencia de Bases , ADN/análisis , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , ARN Mensajero/análisis , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
We have used Northern blot hybridization to study the accumulation of specific cellular mRNAs in Vero cells infected with herpes simplex virus (HSV) type 1 or type 2. HSV-1 infection decreased the cytoplasmic levels of beta- and gamma-actin, beta-tubulin, and histone H3 and H4 mRNAs, though not all at the same rate. HSV-2 infection resulted in a more rapid decrease in actin and histone mRNA levels compared with HSV-1 infection. The turnover rate of each type of mRNA studied was accelerated in HSV-infected cells compared with the rate in uninfected cells. Cellular mRNA degradation was induced by HSV infection under conditions of (i) inhibition of de novo protein synthesis, (ii) inhibition of de novo RNA synthesis, (iii) infection with HSV-1(17) tsK, which fails to produce early and late viral gene products at the nonpermissive temperature, and (iv) infection with purified virions in the presence of actinomycin D. We have concluded that, in Vero cells, cellular mRNA degradation is induced by a factor associated with the infecting HSV virion and thus does not require de novo RNA or protein synthesis. Despite the overall inhibition of cellular mRNA accumulation, a novel 2.2-kilobase cytoplasmic actin transcript was produced in HSV-infected cells when viral gene expression was allowed. The level of accumulation of cytoplasmic host mRNAs was compared with the rate of cellular protein synthesis under different conditions of infection. This analysis suggests that both HSV-1 and HSV-2 require an additional function(s) to completely inhibit cellular protein synthesis.