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PubMed1987

Acclimation of Ribulose Bisphosphate Carboxylase and mRNAs to Changing Irradiance in Adult Tobacco Leaves: Differential Expression in LSU And SSU mRNA.

The transfer of Nicotiana tabacum plants grown in low light (60 micromoles quanta per square meter per second) to higher light (360 micromoles quanta per square meter per second) was previously shown to induce adaptive stimulation of photosynthetic capacities. The variations of ribulose bisphosphate carboxylase/oxygenase (RubisCo) expression in mature leaves was examined as a result of this acclimation. Maximum or initial activities increased markedly after low- to high-light transfer with a maximum effect after 2 to 3 days. The higher activity is mainly explained by RubisCo protein synthesis as shown by immunorocket technique. Small subunits of RubisCo (SSU) mRNA relative content determined by hybridization of total RNA with DNA probe by Dot-blot method, followed the same pattern as RubisCo quantity. The magnitude of this response was amplified when more contrasting light conditions (25 versus 360 micromoles per square meter per second) were established on the same leaf: RubisCo activity, RubisCo protein, and SSU mRNA contents decreased in the shaded zone and increased in the high-light zone within 1 day. After 2 days the shade/light ratio was 1 to 3 for RubisCo protein and 1 to 4 for SSU-RNA, whereas the ratios remained equal to one in controls. Hybridization of the same RNA extracts with large subunits of RubisCo (LSU) probe showed no variation in LSU-RNA content. So in green adult leaves, the expression of SSU and LSU genes is regulated differently. The observed white light quantitative effect on RubisCo expression was not dependent on the photosynthetic rate or assimilate content since low CO(2) concentration around the leaf after the light shift did not modify the response.

Journal Article↗
PubMed2003

[Elevated expression of argentophilic proteins from the nucleolar organizer regions in myocardium of patients with obstructive hypertrophic cardiomyopathy and mutations in p53 tumor suppressor gene].

Silver staining for nucleolar proteins was used to evaluate the ribosomal genes activity in cardiomyocytes (Cm) and fibroblast-like cells (FBS) of intraventricular septum, and other regions of the left ventricle. Specimens to be analysed were taken from 7 patients with idiopathic obstructive hypertrophic cardiomyopathy (OHCM). In this group the quantity of nucleoli and AgNORs, reflecting the transcriptional and processing level of pre-ribosomal RNA in all type of cells, was at least 2 and 3 times higher than in patients with essential hypertension and in healthy control, respectively. We suggest that nucleolar hypertrophy in patients with hypertrophic cardiomyopathies, inappropriate to hypertrophy in other pathological conditions, may be most probably of compensatory character, and this may be in part explained by nuclear hyperploidy and chromosomal endoreduplication. We have noted a marked heterogeneity in shape, size and quantity of nucleoli, and in AgNORs of FBC. Nuclei with modulated phenotype containing nucleoli of high activity were revealed. This article presents the first data on p53 mutation identification in patients with advanced OHCM.

Adult↗
PubMed1996

Binding of a 40-kDa protein to the N-myc 3'-untranslated region correlates with enhanced N-myc expression in human neuroblastoma.

Subclones of neuronal (N) and non-neuronal (S) cells established from neuroblastoma tumors cultured in vitro differ in their growth characteristics and N-myc expression. N (W-N) cells derived from the NBL-W cell line express 5-fold higher levels of N-myc mRNA and 10-12-fold higher levels of protein than S cells (W-S), despite having the same N-myc copy number. This study demonstrates that the steady-state levels of N-myc are largely determined by differences in N-myc mRNA stability. The half-life of N-myc mRNA in the W-N cells is approximately 35 min compared with approximately 6 min in the W-S cells. Turnover of labile mRNAs is thought to be mediated in part by the interactions of trans-acting factors with elements within the 3'-untranslated region. RNA UV cross-linking assays using W-N cell lysate demonstrate abundant quantities of a protein complex that is 40 kDa in size (p40) that binds to the N-myc 3'-untranslated region. p40 is barely detectable in W-S cells. We have mapped two distinct regions within the 3'-UTR that specifically bind p40 (base pairs 5694-5715 and 6465-6482). Analysis of nine additional neuroblastoma cell lines shows that p40 activity correlates with enhanced expression of N-myc. p40 activity is also detected in 5 of 19 primary neuroblastomas, and activity is associated with clinically aggressive disease. In the accompanying study, we identify p40 as a member of the embryonic lethal abnormal vision (ELAV)-like family of RNA-binding proteins. Our studies suggest that ELAV-like proteins may play a role in the regulation of N-myc mRNA turnover and thereby modulate the steady-state levels of N-myc expression and tumor cell phenotype.

Binding Sites↗
PubMed1985

Nucleoprotein autoantibodies in lupus erythematosus.

Autoantibodies against deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) proteins are commonly detected in patients with lupus erythematosus (LE). Antibodies against native DNA are frequently detected in a subset of LE patients with a high prevalence of renal disease. Single-stranded DNA antibodies are also commonly detected in patients with systemic lupus erythematosus (SLE) but recent evidence indicates that approximately 25% of patients with benign, cutaneous (discoid) lupus also possess single-stranded DNA IgM autoantibodies. LE patients also frequently possess antibodies directed against a variety of ribonuclear proteins (RNP). These RNA protein autoantibodies are generally divided into two groups. One group is termed snRNPs (small nuclear ribonuclear protein); the other is termed scRNPs (small cytoplasmic ribonuclear protein). Anti-RNA protein autoantibodies occur as frequently in patients with SLE as do native DNA antibodies. Furthermore, in contradistinction to nDNA antibodies, lupus patients generally make large quantities (detected by gel precipitin techniques) of anti-RNP antibodies. The anti-RNP antibodies are directed against proteins that bind with specific RNA nucleotides. The best evidence at present indicates that these RNA proteins containing the specific RNA nucleotides are involved in RNA processing and post-translational activities such as protein synthesis. Furthermore, these SLE autoantibodies are now being employed, together with other autoantibody systems detected in other connective tissue diseases, to define the biological role of the respective RNA proteins.

Autoantibodies↗
PubMed2006

Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules.

Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function.

APOBEC-3G Deaminase↗
PubMed2001

Focal congenital alveolar proteinosis associated with abnormal surfactant protein B messenger RNA.

Two siblings presented with typical clinical features of congenital pulmonary alveolar proteinosis (PAP). Necropsy of one sibling revealed scattered foci of the diagnostic histologic changes in the lung tissue. In contrast to infantile and adult PAP, focal distribution is uncommon in congenital PAP. Defective expression of the granulocyte-macrophage colony-stimulating factor receptor was ruled out. The surfactant protein B (SP-B) content in the lung tissue of the autopsied patient was low, and a deletion in the SP-B messenger RNA was detected. We speculate that the PAP in our patients was related to the reduced quantity and/or to the altered quality of SP-B.

Chromosome Deletion↗
PubMed1997

Activity of a 40 kDa RNA-binding protein correlates with MYCN and c-fos mRNA stability in human neuroblastoma.

Subclones of neuroblastic (N) and substrate adherent (S) cells have been established from neuroblastoma tumours cultured in vitro which differ in growth characteristics and MYCN expression. N cells derived from the NBL-W cell line (W-N) express 5-fold higher levels of MYCN mRNA and 10-fold higher levels of MYCN protein than S cells (W-S), despite having the same MYCN copy number. In an effort to identify the molecular mechanisms responsible for the disparity in steady-state MYCN levels, the rate of MYCN mRNA degradation was measured in the two subclones. The half-life of MYCN mRNA in the W-N cells was approximately 45 min compared to approximately 6 min in the W-S cells. Similarly, the half-life of another labile mRNA, c-fos, differed in W-N and W-S cells (30 min versus 15 min, respectively). The turnover of labile mRNAs is thought to be mediated by the interactions of trans-acting factors with AU-rich elements within the 3' untranslated region. RNA UV cross-linking assays using W-N cell lysate demonstrated abundant quantities of a protein, 40 kDa in size (p40), that bound specifically to AU-rich elements within the MYCN and c-fos 3' untranslated region. However, p40 was barely detectable in W-S cells. Our studies suggest that p40 may play a role in determining neuroblastoma phenotype by regulating MYCN and c-fos mRNA turnover.

Binding Sites↗
PubMed1981

Thyroid hormones and muscle protein turnover. The effect of thyroid-hormone deficiency and replacement in thryoidectomized and hypophysectomized rats.

We have investigated the effects of thyroidectomy, hypophysectomy and 3,3',5-tri-iodothyronine replacement on protein synthesis and degradation in skeletal muscle in vivo. Thyroidectomy resulted in a decrease in the rate of protein synthesis as a result of a loss of RNA. However, RNA activity, the rate of protein synthesis per unit of RNA, was not decreased. This was the case in both young growing rats and mature nongrowing rats. Tri-iodothyronine treatment of thyroidectomized rats increased protein synthesis by increasing RNA concentration without changes in RNA activity, and this occurred even when food intake was restricted to prevent any increase in growth. The rate of protein degradation was decreased by thyroidectomy and increased by tri-iodo-thyronine replacement in both animals fed ad libitum and food-restricted animals. Hypophysectomy decreased protein synthesis by decreasing both RNA concentration and activity. these changes were reversed by tri-iodothyronine treatment even in the presence of persistent marked hypoinsulinaemia. This indicates that tri-iodothyronine can activate athe translational phase of protein synthesis in muscle in the absence of significant quantities of insulin. However, tri-iodothyronine does not seem to be obligatory for the maintenance of normal RNA activity in muscle, since in the thyroidectomized rat, in which plasma insulin concentrations are normal, RNA activity is maintained. From a consideration of the magnitude of changes in RNA activity observed in these experiments, it would appear that alterations in rates of elongation as well as initiation are involved in the changes in RNA activity.

Animals↗
PubMed2000

Mechanisms for genetically predetermined differential quantitative expression of HLA-A and -B antigens.

Previous studies showed that different HLA-A and -B antigens are differentially expressed in cells. Their relative quantities are genetically predetermined and inherited according to Mendelian laws. To investigate mechanisms responsible for this differential expression, a correlation study between the relative quantities of different HLA-A and -B proteins and their mRNA levels in eight different HLA-phenotyped lymphoblastoid cell lines (LCLs) were performed. The results show proportional correlation in all the studied cell lines except those that are positive for HLA-A24. Study of the turnover of HLA antigens reveals that different HLA-A and -B antigens are proportionally degraded. Measurement of the relative quantities of HLA-A and -B mRNAs in six LCLs before and after treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of RNA polymerase II, demonstrates that HLA-A and -B mRNAs are proportionally degraded except slight differences in two LCLs. Measurement of the relative quantities of different HLA-A and -B pre-mRNAs in nuclei shows that they are not proportional to the relative quantities of their respective mature mRNAs in cytoplasm in four of six LCLs. These results indicate that combinations of different regulatory steps which include gene transcription, pre-mRNA splicing and mRNA degradation are involved in the genetically predetermined quantitative differential expression of HLA-A and -B antigens. Transcription of HLA genes and splicing of HLA pre-mRNAs appear to be the dominant regulatory steps.

Cell Line, Transformed↗
PubMedDate not supplied

RNA transcripts of hepatitis B virus in hepatocellular carcinoma.

The states of hepatitis B virus DNA, RNA transcripts and antigens (HBsAg and HBcAg) were studied in the liver with hepatocellular carcinoma by blot hybridization and immunohistological methods. We used whole hepatitis B virus DNA and gene specific probes (S, C and X genes) for hybridization. Integrated viral DNA was found in all five tumors, but episomal DNA was not detected in the neoplastic tissue. In the nonneoplastic cirrhotic liver, episomal DNA was found in four cases and integrated viral DNA in four cases. A large amount of hepatitis B virus-specific RNA transcripts was demonstrated in nonneoplastic liver of all five cases. Two major transcripts, 2.4 and 3.4 kb in length, were identified. The former hybridized with the S gene probe and the latter with the C gene probe, suggesting that they were messenger RNAs for HBsAg and HBcAg, respectively. In contrast to nonneoplastic liver, RNA transcripts were found in the neoplastic portion in only two cases, small in quantity; they primarily hybridized with S and X, but not with C genes. Novel species of 4.0 and 3.9 kb transcripts were found in the neoplastic and the nonneoplastic portions, respectively, in one case. They may represent fusion transcripts of integrated viral DNA and cellular flanking sequences.

Adult↗
PubMed1977

Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody.

A procedure is presented for the purification of specific mRNAs, which exploits the ability of antibodies prepared against a native protein to bind to the nascent polypeptide on the polysome. Rather than precipitating these soluble antibody-polysome complexes with anti-antibody, which can lead to nonspecific trapping of polysomes, we have linked the anti-antibody to an insoluble matrix. Thus, the antibody-polysome complex binds to the anti-antibody support and nonspecific polysomes can easily be removed by several washes. We have found para-aminobenzyl cellulose (PAB cellulose), to be a suitable matrix for this purpose. This support can bind large quantities of anti-antibody and it displayed no detectable nonspecific affinity for polysomes or RNA. Using this procedure, we have obtained an apparently homogeneous preparation of ovalbumin mRNA.

Animals↗
PubMed1981

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ.

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH(4))(2)SO(4) precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.

Journal Article↗
PubMed2003

The tRNA-like domains of E coli and A.aeolicus transfer-messenger RNA: structural and functional studies.

Transfer-messenger RNA (tmRNA, 10Sa RNA or ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The understanding at molecular level of this ubiquitous quality control system in eubacteria requires structural information. Here, we describe the purification and structural analysis of a functional fragment of both Aquifex aeolicus and Escherichia coli tmRNA, recapitulating their tRNA-like domain, which were expressed in vivo from synthetic genes. Both recombinant RNA are correctly processed at both 5' and 3' ends and are produced in quantities suitable for structural analysis by NMR and/or X-ray crystallography. The sequence and solution structure of the tRNA-like domains were analysed by various methods including structural mapping with chemical and enzymatic probes and 2D NMR spectroscopy. The minimalist RNAs contain two post-transcriptional base modifications, 5-methyluridine and pseudouridine, as the full-length tmRNA. Both RNAs fold into three stems, a D-analogue, a T-loop and a GAAA tetra-loop. 2D NMR analysis of the imino proton resonances of both RNAs allowed the assignment of the three stems and of a number of tertiary interactions. It shows the existence of interactions between the TPsiC-loop and the D-analogue, exhibiting a number of similarities and also differences with the canonical tRNA fold, indicating that RNA tertiary interactions can be modulated according to the sequence and secondary structure contexts. Furthermore, the E.coli minimalist RNA is aminoacylatable with alanine with a catalytic efficiency an order of magnitude higher than that for full-length tmRNA.

Alanine↗
PubMed1981

Hydra viridis: transfer of metabolites between Hydra and symbiotic algae.

"Back transfer" of metabolites from food to endosymbiotic algae in the digestive cells of Hydra viridis was demonstrated. Brine shrimp nauplii labeled with tritiated precursors of protein and nucleic acids (DNA and RNA) were fed to light and dark grown hydras. The fate of the label after a single feeding with radioactive material in hydra and algal fractions was followed by scintillation counting and autoradiographic techniques. Labeled thymidine was incorporated into DNA in both light- and dark-grown hydras. Although the symbiosis persists indefinitely in hydras in darkness (7-10 days) the number of algae per cell is reduced. Tritiated orotic acid and tritiated uridine, RNA precursors, were incorporated into peptides and proteins, and to a lesser extent into simple sugars, oligosaccharides, and oligonucleotides in hydra and algal fractions. Thus the metabolites of the brine shrimp food are available to both partners. A decrease over time in label introduced as 3H-orotic acid and 3H-uridine and incorporated into hydra RNA is compensated for by an increase in label in the algae, implying competition for constant quantities of metabolites from the single feeding. Although food availability, light, number of algae per cell, and other factors influence the quantity and rate of nutrient transfer between the partners, in both light and dark grown hydras the amount of "back transfer" of metabolites to the symbiotic algae is impressive.

Animals↗
PubMed1988

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli.

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.

Bacterial Proteins↗
PubMed1997

Relative levels of alpha-, beta-, and gamma-mRNA from patients with severe and intermediate beta-thalassemia major.

We have determined the relative quantities of gamma- and beta-mRNAs and the alpha/beta-mRNA ratios in 37 patients with beta-thalassemia major with specific genotypes, namely 8 with a homozygosity for codon (CD) 39 (C-->T), 7 with a homozygosity for IVS-I-110 (G-->A), 5 with a homozygosity for IVS-I-6 (T-->C), for 15 patients with compound heterozygosities for 2 of these 3 mutations, and for 2 patients with the IVS-I-110 (G-->A)/-87 (C-->G) mutations. None had an alpha-thalassemia. Twelve patients had thalassemia intermedia and the remainder, transfusion-dependent severe conditions. Differences in phenotype were observed for compound heterozygotes involving the IVS-I-6 (T-->C) mutation in combination with either the IVS-I-110 (G-->A) or the CD 39 (C-->T) mutations: patients with thalassemia intermedia had a lower alpha/beta-mRNA ratio, about half of that of the patients with severe beta-thalassemia major. This might suggest a higher beta-mRNA synthesis in some patients than in others with the same genotype; mutations in promoter, enhancer, and/or locus control region sequences may be responsible for these differences. In vitro chain synthesis data were too incomplete to be helpful in this study. The RT-PCR procedure allowed the separation of abnormal (extended) mRNA from normal beta-RNA in subjects carrying the IVS-I-110 (G-->T) mutation. The relative quantities of this beta Th-mRNA (% of beta A + beta Th) were determined by scanning of the appropriate autoradiograms; they averaged 25% for homozygotes and about 4% for heterozygotes, indicating a considerable instability of the message.

Codon↗
PubMed1999

Mutation rates among RNA viruses.

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of micro(g) approximately 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population.

Humans↗
PubMed1976

[Polyadenylic acid rich RNA fractions in primary hepatomas induced with diethylnitrosamine].

The column chromatography with unmodified cellulose revealed a polyadenilic acid rich fraction of total RNA from diethylnitrosamine induced primary hepatomas and normal rat liver. It was shown that the quantity of this fraction was increased to 3.15 per cent. The 2.5% polyacrylamide gel electrophoresis of the polyadenylic acid rich fraction indicated its different spectra in tumors and normal rat liver, the main change being in bipolymer fractions with sedimentation coefficient 28 S and higher.

Animals↗