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PubMed1985

Chymotrypsin treatment of glucocorticoid receptor attenuates RNA-dependent inhibition of DNA binding. Evidence for a distinct RNA-binding site.

Certain types of RNA can prevent the association of the rat liver glucocorticoid receptor with DNA. This inhibition of receptor binding to DNA cannot be mitigated by increasing amounts of DNA, suggesting that the RNA is not merely acting as a competitive inhibitor. Treatment of partially purified receptor with low concentrations of chymotrypsin eliminates the inhibitory effects of some RNAs without negatively affecting the DNA-binding ability of the receptor. Potent inhibitors of the receptor-DNA association, such as poly(G) and poly(X), still inhibit DNA binding of the treated receptor, although to a lesser extent than the untreated controls. However, moderate inhibitors, such as tRNA and poly(U), no longer inhibit the receptor-DNA association at low concentrations. We take these findings to suggest that RNA inhibition of DNA binding is due to the interaction of the RNA at a distinct RNA-binding site. This site may serve as a regulator site for the receptor-DNA association.

Animals↗

PubMed2004

A distinctive ribonuclease from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum.

A ribonuclease with an N-terminal sequence distinct from other mushroom ribonucleases was isolated from fresh fruiting bodies of the medicinal mushroom Ganoderma lucidum. The ribonuclease was adsorbed on DEAE-cellulose and Q-Sepharose, and unadsorbed on CM-Sepharose. It possessed a molecular mass of 42 kDa as judged by gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molecular mass was similar to that of straw mushroom ribonuclease but much higher compared with those of other mushroom ribonucleases. The ribonuclease was unique among mushroom ribonucleases in that it exhibited the highest potency toward poly(U), followed by poly(A). Its activity toward poly(G) and poly(C) was about one-half of that toward poly(A) and one-quarter of that toward poly(U). A pH of 4.0 and a temperature of 60 degrees C were required for optimal activity of the enzyme. The optimum pH was low compared with those reported for other mushroom ribonucleases.

Agaricales↗

PubMed2004

A ribonuclease with antimicrobial, antimitogenic and antiproliferative activities from the edible mushroom Pleurotus sajor-caju.

A 12 kDa ribonuclease preferential for poly U and with much lower activity toward poly A, poly G and poly C was isolated from fresh fruiting bodies of the mushroom Pleurotus sajor-caju. A purification procedure involving ion exchange chromatography on CM-cellulose, affinity chromatography on Red-Sepharose and Heparin-Sepharose, and fast protein liquid chromatography-gel filtration on Superdex 75 was used. The ribonuclease was adsorbed on all of the first three types of chromatographic media. It exhibited some activity toward herring sperm DNA and calf thymus DNA. The ribonuclease activity was unaffected in the presence of KCl (10 and 100 mM) and NaCl (100 mM and 1 M), but was strongly inhibited by CuSO4 (0.01 and 0.1 mM) and less potently inhibited by other divalent salts including MgCl2, CaCl2, ZnCl2, ZnSO4 and FeSO4. The optimal pH was 5.5 and the ribonuclease was stable up to 60 degrees C for 1 h. The ribonuclease inhibited mycelial growth in the fungi Fusarium oxysporum and Mycosphaerella arachidicola with an IC50 value of 95 and 72 microM, respectively. Out of the 12 species of bacteria tested, only Pseudomonas aeruginosa and Staphylococcus aureus were inhibited in growth by the ribonuclease. Viability of the tumor cells HepG2 (hepatoma) and L1210 (leukemia) was reduced with an IC50 of 0.22 and 0.1 microM, respectively in the presence of the ribonuclease. The ribonuclease inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 158 nM and 3H-methyl-thymidine uptake by murine splenocytes with an IC50 of 65 nM.

Animals↗

PubMed2003

A ubiquitin-like peptide with ribonuclease activity against various polyhomoribonucleotides from the yellow mushroom Cantharellus cibarius.

A peptide, with a molecular weight of 9K and an N-terminal sequence identical to that of ubiquitin, was isolated from fresh fruiting bodies of the yellow mushroom Cantharellus cibarius. The peptide manifested ribonucleolytic activity toward poly A, poly C, poly G, and poly U, with the activity toward the first two polyhomoribonucleotides being higher. Maximal activity toward yeast tRNA was observed at a temperature of 70 degrees C and a pH of 7. The peptide was adsorbed on DEAE-cellulose, CM-Sepharose, and Q-Sepharose. The yield of the peptide was 7mg from 1.8kg mushroom.

Amino Acids↗

PubMed2000

Ribonuclease, cell-free translation-inhibitory and superoxide radical scavenging activities of the iron-binding protein lactoferrin from bovine milk.

The purpose of this study was to characterize the ribonuclease (RNase) and cell-free translation-inhibitory activities of lactoferrin isolated from bovine milk. It was found that bovine lactoferrin exhibited ribonucleolytic activity toward yeast transfer RNA in a dose-dependent manner. The pH optimum for this RNase activity was in the vicinity of 7.5. Lactoferrin exerted RNase activity on poly C with an activity of 2.15 U/mg. No activity was detected toward poly A, poly G, and poly U. The milk protein inhibited cell-free translation in rabbit reticulocyte lysate with an IC50 of 9.6 microM. The protein was devoid of N-glycosidase activity characteristic of ribosome inactivating proteins which also possess RNase and cell-free translation-inhibitory activities. It inhibited superoxide radical formation.

Animals↗

PubMed2006

Selective photoelectrochemical detection of DNA with high-affinity metallointercalator and tin oxide nanoparticle electrode.

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-10) M in solution.

DNA↗

PubMedDate not supplied

Separation technique for messenger RNAs by use of schizophyllan/poly(A) tail complexation.

Schizophyllan (SPG) is one of the water soluble beta-1,3-glucans and has a peculiar molecular recognition capability, namely, the single stranded SPG (s-SPG) can form a stoichiometric complex with certain polynucleotides such as poly(C) and poly(A), although it cannot bind poly(G) and poly(dC) at all. In this paper, we prepared an s-SPG-appended column and made an attempt to separate polynucleotides on the bases of this molecular recognition capability. The s-SPG-appended column trapped only such RNAs that could form the complex with s-SPG but eluted other RNAs which did not form the complex. Encouraged by the results in the model system, we extended the s-SPG-appended column into separation of native messenger RNAs (mRNAs) from a RNA mixture (total RNA) obtained from yeast. Since eukaryotic mRNAs have a poly(A) tail with 150-300 bases, we supposed that the tails would be trapped by the s-SPG-appended column. The results indicate that mRNAs were separated from total RNA in good yield and with high purity. It should be emphasized that this is the first device to separate natural mRNAs without using a dA/dT Watson-Crick-type interaction.

Carbohydrate Sequence↗

PubMed2007

Polyadenylation proteins CstF-64 and tauCstF-64 exhibit differential binding affinities for RNA polymers.

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, tauCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between tauCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of tauCstF-64 and CstF-64 have different affinities for RNA elements. We quantified K(d) values of CstF-64 and tauCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than tauCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal alpha-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is mportant in RNA sequence recognition. This supports the hypothesis that tauCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements.

Amino Acid Sequence↗

PubMed2003

The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.

Animals↗

PubMed1999

The nucleoporin nup153 plays a critical role in multiple types of nuclear export.

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin beta to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin alpha/beta or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.

Animals↗

PubMed1991

Nucleic acid-binding specificities of tobacco chloroplast ribonucleoproteins.

Many ribonucleoproteins (RNPs) are involved in the regulation of gene expression at the post-transcriptional level. We previously isolated three nuclear-encoded RNPs from tobacco chloroplasts. Here we report their binding specificities for various nucleic acids. The three RNPs synthesized in vitro were subjected to binding assays using ssDNA, dsDNA and four ribonucleotide homopolymers. The affinities for ribonucleotide homopolymers are higher than those for ssDNA and dsDNA, suggesting that they bind preferentially to RNA in vivo. All three RNPs show high specificities for poly(G) and poly(U) in the presence of 1.2-1.8 M NaCl, providing additional evidence for the similarity to HeLa hnRNP proteins. Possible involvement of these RNPs in the post-transcriptional regulation of chloroplast gene expression is discussed.

Base Sequence↗

PubMed1994

Chicken MAR binding protein p120 is identical to human heterogeneous nuclear ribonucleoprotein (hnRNP) U.

We have previously identified two proteins from chicken oviduct nuclei that specifically bind to matrix/scaffold attachment regions (MARs/SARs). Here one of these proteins, named p120 due to its apparent molecular weight, is purified to near homogeneity and shown to be identical to a previously described component of heterogeneous nuclear ribonucleoprotein particles, hnRNP U, on the basis of amino acid sequence analysis of tryptic peptides. p120 binds to multiple MAR fragments provided they have a minimal length of approximately 700 bp. Binding of MAR fragments is specifically competed by homoribopolymers poly(G) and poly(I), which form four-stranded structures. Our results suggest that p120/hnRNP U may serve a dual function, first as a component of hnRNP particles, and second as an element in the higher-order organization of chromatin.

Amino Acid Sequence↗

PubMed2001

The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation.

The major mRNA degradation pathway involves deadenylation of the target molecule followed by decapping and, finally, 5'-->3' exonuclease digestion of the mRNA body. While yeast factors involved in the decapping and exonuclease degradation steps have been identified, the nature of the factor(s) involved in the deadenylation step remained elusive. Database searches for yeast proteins related to the mammalian deadenylase PARN identified the Pop2 protein (Pop2p) as a potential deadenylase. While Pop2p was previously identified as a factor affecting transcription, we identified a non-canonical RNase D sequence signature in its sequence. Analysis of the fate of a reporter mRNA in a pop2 mutant demonstrates that Pop2p is required for efficient mRNA degradation in vivo. Characterisation of mRNA degradation intermediates accumulating in this mutant supports the involvement of Pop2p in mRNA deadenylation in vivo. Similar phenotypes are observed in yeast strains lacking the Ccr4 protein, which is known to be associated with Pop2p. A recombinant Pop2p fragment encompassing the putative catalytic domain degrades poly(A) in vitro demonstrating that Pop2p is a nuclease. We also demonstrate that poly(A) is a better competitor than poly(G) or poly(C) of the Pop2p nuclease activity. Altogether, our study indicates that Pop2p is a nuclease subunit of the yeast deadenylase and suggests that Pop2p homologues in other species may have similar functions.

Amino Acid Sequence↗

PubMed1980

The translocation inhibitor tuberactinomycin binds to nucleic acids and blocks the in vitro assembly of 50S subunits.

Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.

Binding Sites↗

PubMed1998

Cloning and characterization of a cDNA encoding a novel heterogeneous nuclear ribonucleoprotein-like protein and its expression in myeloid leukemia cells.

We isolated a cDNA encoding a novel heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein on DNA affinity screening of a K562 cDNA expression library with an oligodeoxynucleotide (JKT41) derived from intron 9 of the human myeloperoxidase gene. The cDNA has a 1,305 bp sequence that encodes a polypeptide of 301 amino acid residues. The protein, named JKTBP, contains two repeats of a putative RNA binding domain (RBD), each composed of canonical RNP-2 and RNP-1 motifs, and a glycine- and tyrosine-rich carboxyl terminus. The sequences of these two repeats are highly homologous with those of the 2 x RBD-Gly rich group of hnRNPs. Northern blotting showed that two mRNAs of approximately 1.4 and 2.8 kb were present in most cultured cells examined. The recombinant protein expressed in Escherichia coli interacted with the double-stranded form of JKT41 as well as with its single-stranded form. This interaction was competitively inhibited by the same unlabeled JKT41 and to nearly the same extent by unrelated oligonucleotides. Moreover, the recombinant protein interacted with poly(G) and poly(A), but not with poly(U) or poly(C). Transient expression of the protein in SKM-1 cells repressed the expression of chloramphenicol acetyltransferase reporter genes located downstream of the intron 9 element of JKT41 or intron 7 element of FERE27. The implications of the protein in the biogenesis of mRNA are discussed.

Amino Acid Sequence↗

PubMed1987

Isolation and characterization of an activator for Azotobacter vinelandii nicotinamide mononucleotide glycohydrolase.

Azotobacter vinelandii NMN glycohydrolase [EC 3.2.2.14] has been shown to require absolutely GTP or a high-molecular-weight and heat-stable component for its function. The intracellular activator could be purified from its sonicate by heat treatment, acetone precipitation, phenol extraction, and acid precipitation in a good yield. The purified activator showed high affinity and effectiveness for NMN glycohydrolase (KA = 0.012 optical density unit at 257 nm/ml; Vmax standardized by the activity at 1 mM GTP = 88%). Negative cooperativity of the enzyme activation with the activator was also shown. On treatment with either micrococcal nuclease or pancreatic RNase, the activator activity was completely abolished, whereas pronase and trypsin had no effect. The activator could be replaced by yeast RNA as well as calf liver RNA, whereas DNAs purified from Micrococcus lysodeikticus, T 7 and calf thymus had no effect on the enzyme. Furthermore, poly(G) and poly(I) could function as activators with the same effectiveness as the purified activator, and the enzyme activation with these RNA homopolymers was inhibited by poly(C), suggesting that the activation mechanism is specific with respect to base composition. Based on a kinetic analysis of the enzyme activation with commercial RNAs, together with the results from enzymatic digestion, specific inhibition of the enzyme by spermine, and its chemical properties, the activator was identified as an RNA. A model is described for NMN glycohydrolase regulation in which the RNA activator plays an important role in the NMN salvage cycles.

Azotobacter↗

PubMed1976

Purification and properties of an alkaline ribonuclease from the hepatic cytosol fraction of bullfrog, Rana catesbeiana.

In the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4.22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaC1. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of p-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with p-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column. The molar weight of RNase was determined to be approximately 12,000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNase-RNase inhibitor complex was 130,000. The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2',3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.

Amino Acids↗

PubMed1975

Three distinct forms of nuclear poly(A) polymerase.

Poly(A) polymerase activities have been solubilized from rat liver nuclei and purified by chromatography on Bio-Gel A-1.5M, DEAE-Sephadex and CM-cellulose. Three distinct forms of nuclear poly(A) polymerase have been resolved by chromatography on CM-cellulose. According to their sequence of elution from CM-cellulose these enzyme activities have been termed A, B and C. Enzymes A and B are Mn2+ -dependent, enzyme C requires Mg2+. With the same chromatographic step on CM-cellulose the Mn+ -dependent poly(A) polymerase activities were separated from a Mn2+ -dependent enzyme system capable of synthesizing RNA-primed poly(U), poly(G) and poly(C). The effect of different nuclear and cytoplasmic RNA primers on the rate of poly(A) formation suggests enzyme A to be responsible for the elongation of preexisting poly (A) chains. The phosphorylated derivated derivative of cordycepin, 3'-deoxyadenosine 5'-triphosphosphate (3'-dATP), which is known to inhibit nuclear poly(A) synthesis in vivo, also impairs poly(A) formation in vitro. It is shown that 3'-dATP very probably is not incorporated into poly(A)invitro, suggesting that 3'-dATP primarily affects the catalytic activities of the poly(A) polymerase species rather than directly blocking chain elongation.

Adenosine Triphosphate↗