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PubMed2006

Genetic changes in hepatitis delta virus from acutely and chronically infected woodchucks.

A woodchuck-derived hepatitis delta virus (HDV) inoculum was created by transfection of a genotype I HDV cDNA clone directly into the liver of a woodchuck that was chronically infected with woodchuck hepatitis virus. All woodchucks receiving this inoculum became positive for HDV RNA in serum, and 67% became chronically infected, similar to the rate of chronic HDV infection in humans. Analysis of HDV sequences obtained at 73 weeks postinfection indicated that changes had occurred at a rate of 0.5% per year; many of these modifications were consistent with editing by host RNA adenosine deaminase. The appearance of sequence changes, which were not evenly distributed on the genome, was correlated with the course of HDV infection. A limited number of modifications occurred in the consensus sequence of the viral genome that altered the sequence of the hepatitis delta antigen (HDAg). All chronically infected animals examined exhibited these changes 73 weeks following infection, but at earlier times, only one of the HDV carriers exhibited consensus sequence substitutions. On the other hand, sequence modifications in animals that eventually recovered from HDV infection were apparent after 27 weeks. The data are consistent with a model in which HDV sequence changes are selected by host immune responses. Chronic HDV infection in woodchucks may result from a delayed and weak immune response that is limited to a small number of epitopes on HDAg.

Acute Disease↗
PubMed1986

The gene for the Mr 10,000 phosphoprotein associated with photosystem II is part of the psbB operon of the spinach plastid chromosome.

We present the nucleotide and derived amino acid sequence of the gene for the 10 kd phosphoprotein associated with photosystem II. This gene was identified by comparing the recently published first nine amino acid residues for the 10 kd phosphoprotein of spinach (Farchaus and Dilley 1986) with available sequence data from the spinach plastid chromosome. The gene, designated psbH, is part of an operon that encodes the 51 kd chlorophyll a apoprotein of the photosystem II reaction center (psbB), the phosphoprotein (73 codons), cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in the order given. Northern blot analysis revealed a complex in vivo RNA pattern for this DNA segment resulting from an extensive modification of a 5.6 kb long putative primary transcript which includes a monocistronic RNA species for the phosphoprotein of approximately 420 bases. The deduced amino acid sequence for the phosphoprotein indicates a polypeptide corresponding to a molecular weight of 7.8 kb. Secondary structure predictions place all potential phosphorylation sites on the stromal side of the membrane.

Amino Acid Sequence↗
PubMed1976

Activation of chromatin by acetylation of histone side chains.

The ability of calf thymus chromatin to support DNA-dependent RNA synthesis is markedly increased by modification of the histones with acetic anhydride. The extent of chemical acetylation of histones H2a, H2b, H3, and H4 required for the increase of template activity of the chromatin is within that which can be achieved enzymatically in the cell. The evidence suggests that the acetylation of histone side chains stimulates the rate of chain elongation during transcription of chromatin.

Acetylation↗
PubMed2003

RNA-modifying enzymes.

A bewildering number of post-transcriptional modifications are introduced into cellular RNAs by enzymes that are often conserved among archaea, bacteria and eukaryotes. The modifications range from those with well-understood functions, such as tRNA aminoacylation, to widespread but more mysterious ones, such as pseudouridylation. Recent structure determinations have included two types of RNA nucleobase modifying enzyme: pseudouridine synthases and tRNA guanine transglycosylases.

Binding Sites↗
PubMed1997

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II.

5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.

Amino Acid Sequence↗
PubMed2006

Synthesis, oxidation behavior, crystallization and structure of 2'-methylseleno guanosine containing RNAs.

We have recently introduced a basic concept for the combined chemical and enzymatic preparation of site-specifically modified 2'-methylseleno RNAs which represent useful derivatives for phasing of X-ray crystallographic data during their three-dimensional structure determination. Here, we introduce the first synthesis of an appropriate guanosine phosphoramidite, which complements the thus far established set of 2'-methylseleno-modified uridine, cytidine, and adenosine building blocks for solid-phase synthesis. The novel building block was readily incorporated into RNA. Importantly, it was the 2'-methylseleno-guanosine-labeled RNA that allowed us to reveal the reversible oxidation/reduction behavior of the Se moiety and thus it represents a valuable contribution to the understanding of the action of threo-1,4-dimercapto-2,3-butanediol (DTT) required during solid-phase synthesis, deprotection, and crystallization of selenium-containing RNA. In addition, we investigated 2'-methylseleno RNA with respect to crystallization properties. Our studies revealed that the Se modification significantly increases the range of conditions leading to crystal growth. Moreover, we determined the crystal structures of model RNA helices and showed that the Se modification can affect crystal packing interactions, thus potentially expanding the possibilities for obtaining the best crystal form.

Crystallization↗
PubMed1995

In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.

Aldehydes↗
PubMed2005

Recognition of RNA by amide modified backbone nucleic acids: molecular dynamics simulations of DNA-RNA hybrids in aqueous solution.

Thermodynamic and structural properties of a chemically modified DNA-RNA hybrid in which a phosphodiester linkage is replaced by a neutral amide-3 linkage (3'-CH(2)-CONH-5') were investigated using UV melting experiments, molecular dynamics simulations in explicit water, and continuum solvent models. van't Hoff analysis of the experimental UV melting curves suggests that the significant increase of the thermodynamic stability of a 15-mer DNA-RNA with seven alternated amide-3 modifications (+11 degrees C) is mainly due to an increased binding enthalpy. To further evaluate the origin in the observed affinities differences, the electrostatic contribution to the binding free energy was calculated by solving the Poisson-Boltzmann equation numerically. The nonelectrostatic contribution was estimated as the product of a hydrophobic surface tension coefficient and the surface area that is buried upon double strand formation. Structures were taken from 10 ns molecular dynamics simulations computed in a consistent fashion using explicit solvent, counterions, and the particle-mesh Ewald procedure. The present preliminary thermodynamic study suggests that the favorable binding free energy of the amide-3 DNA single strand to the complementary RNA is equally driven by electrostatic and nonpolar contributions to the binding compared to their natural analogues. In addition, molecular dynamics simulations in explicit water were performed on an amide-3 DNA single strand and the corresponding natural DNA. Results from the conformations cluster analysis of the simulated amide-3 DNA single strand ensembles suggest that the 25% of the population sampled within 10 ns has a pre-organized conformation where the sugar C3' endo pucker is favored at the 3'-flanking nucleotides. These structural and thermodynamic features contribute to the understanding of the observed increased affinities of the amide-3 DNA-RNA hybrids at the microscopic level.

Amides↗
PubMed1997

Modulation of RNase H activity by modified DNA probes: major groove vs minor groove effects.

We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups. The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes were formed using DNA probes designed to deliver metal complexes via either the major groove or the minor groove of the duplex. The duplexes were treated with Escherichia coli RNase H. Modifications in the major groove produced the same RNA cleavage pattern as unmodified DNA probes. However, minor groove substituents inhibited RNA cleavage over a four-base region. Comparison was made with a DNA probe containing a 2'-OMe modification. Our results support enzyme binding in the minor groove of a DNA/RNA duplex. We do not observe cleavage directly across from the modified nucleoside. The RNA cleavage efficiency effected by RNase H and a DNA probe decreases as follows: unmodified DNA > or = C-5 modified DNA >> c2'-modified DNA > C1'-modified DNA. Results with 28-mer RNA substrates roughly parallel those obtained with a 159-mer RNA target. The differences observed between low and high MW RNA substrates can be explained by a much higher enzyme-substrate binding constant for the high MW target.

Base Sequence↗
PubMed2006

IFN-beta-induced SOCS-1 negatively regulates CD40 gene expression in macrophages and microglia.

Costimulation between T cells and antigen-presenting cells is required for adaptive immune responses. CD40, a costimulatory molecule, is expressed in macrophages and microglia. The aberrant expression of CD40 is involved in human diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease. CD40 expression is induced by a variety of stimuli, including IFN-gamma and lipopolysaccharide (LPS). In this study, we describe the molecular basis by which IFN-beta, a cytokine with immunomodulatory properties, regulates CD40 gene expression. IFN-beta induces CD40 expression in macrophages and microglia at the transcriptional level, and GAS elements in the CD40 promoter are required for IFN-beta-induced CD40 promoter activity. The critical role of signal transducers and activators of transcription-1alpha (STAT-1alpha) in this response was confirmed by utilizing primary microglia from STAT-1alpha deficient mice. IFN-beta induces suppressor of cytokine signaling-1 (SOCS-1) gene expression, which inhibits cytokine signaling by inhibiting activation of STAT proteins. The ectopic expression of SOCS-1 abrogates IFN-beta-mediated STAT-1alpha activation and inhibits IFN-beta-induced CD40 expression. IFN-beta-induced recruitment of STAT-1alpha and RNA Pol II and permissive histone modifications on the CD40 promoter are also inhibited by SOCS-1 overexpression. These novel results indicate that IFN-beta-induced SOCS-1 plays an important role in the negative regulation of IFN-beta-induced CD40 gene expression.

Animals↗
PubMed2004

Centromere-encoded RNAs are integral components of the maize kinetochore.

RNA is involved in a variety of chromatin modification events, ranging from large-scale structural rearrangements to subtle local affects. Here, we extend the evidence for RNA-chromatin interactions to the centromere core. The data indicate that maize centromeric retrotransposons (CRMs) and satellite repeats (CentC) are not only transcribed, but that nearly half of the CRM and CentC RNA is tightly bound to centromeric histone H3 (CENH3), a key inner kinetochore protein. RNAs from another tandem repeat (180-bp knob sequence) or an abundant euchromatic retroelement (Opie) are undetectable within the same anti-CENH3 immune complexes. Both sense and antisense strands of CRM and CentC, but not small interfering RNAs homologous to either repeat, were found to coimmunoprecipitate with CENH3. The bulk of the immunoprecipitated RNA ranged in size from 40 to 200 nt. These data provide evidence for a pool of protected, single-stranded centromeric RNA within the centromere/kinetochore complex.

Base Sequence↗
PubMed1985

Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent.

The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions. In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions. In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination. These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity. However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10. These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly. Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions.

Alkylation↗
PubMed1999

Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis.

Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site were used as tools to study the cleavage mechanism of RNase P RNA from Bacillus subtilis. Both the Sp and the Rp-diastereomer reduced the rate of processing at least 10(4)-fold under conditions where the chemical step is essentially rate-limiting. Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground state binding to B. subtilis RNase P RNA. Processing of the Rp-diastereomeric ptRNA could be restored in the presence of Mn2+or Cd2+, demonstrating direct metal ion coordination to the pro -Rp oxygen during catalysis. With Cd2+, processing required the presence of another metal ion, such as Ca2+or Mg2+, to mediate substrate binding. This is in contrast to Escherichia coli RNase P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presence of Cd2+as the sole divalent metal ion. Analysis of [Cd2+]-dependent processing of the Rp-diastereomeric substrate by B. subtilis RNase P RNA was consistent with the involvement of at least two metal ions in catalysis. The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+on cleavage of unmodified ptRNA. In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+nor Cd2+were able to restore significant cleavage at this location. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Unaffected ground state binding of the Sp-diastereomeric ptRNA but a >/=10(4)-fold reduced hydrolysis rate may indicate a crucial role of the pro -Sp oxygen in transition state stabilization or may be attributed to steric exclusion of catalytic metal ions. Based on our comparative analyses of B. subtilis and E. coli RNase P RNA, each representing the main structural subtypes of bacterial RNase P RNA, common features in terms of active site constraints and role of catalytic metal ions can now be formulated for bacterial RNase P RNAs. On the other hand, substantial and unexpected differences with respect to the overall metal ion requirements and tRNA binding modes have been observed for the two catalytic RNAs.

Bacillus subtilis↗
PubMed2002

Proteomics in studies of signal transduction in epithelial cells.

An understanding of the complexity of cancer is important for correct diagnostics and efficient treatment of this disease. Recent developments of proteomics technologies allow us to address the complexity of tumorigenesis at a level of global protein profiling. This review discusses recent studies of signaling processes in cells of epithelial origin undertaken with the use of global protein profiling. Tumors of epithelial origin comprise about 90% of human breast cancers, and it is believed that transformation of breast epithelial cells shares common features of transformation with other mammalian cells: destabilization of the genome followed by acquisition of immortalization, unrestricted growth, evasion of death-inducing signals, and acquisition of invasive and tumor promoting characteristics. Functional proteomics of growth-promoting, growth-inhibiting, and pro-apoptotic signaling pathways, in combination with proteomics studies of breast epithelial cell differentiation and profiling of breast tumorigenesis, revealed groups of regulated proteins: structural components, stress-regulated proteins, regulators of transcription, translation and RNA processing, and regulators of posttranslational modifications, e.g., kinases, phosphatases, and proteases. The first lesson of proteomics studies is the discovery of significant number of new targets, as compared to total number of affected proteins. The second lesson is the poor correlation between expressions of proteins and their mRNAs. The third lesson is the low amplitude of protein changes compared to that observed for mRNA. These observations also recommend the analysis of signaling patterns rather than separate signaling pathways.

Animals↗
PubMed1982

Molecular biology of rotaviruses. IV. Molecular cloning of the bovine rotavirus genome.

A new cloning strategy has been developed for cloning the genomes of double-stranded RNA viruses by using bovine rotavirus as a test system. The major modification adopted was the use of denatured polyadenylated double-stranded RNA as the template for reverse transcriptase. This allowed the two complementary strands of cDNA to be synthesized in a single reaction and removed the need for S1 nuclease digestion to remove the 5' hairpin structure normally generated in cDNA synthesis.

Animals↗
PubMed1998

Conformational study of DNA-RNA duplexes containing MMI substituted phosphodiester linkages by FTIR spectroscopy.

Six methylene(methylimino) (MMI, Bhat et al. J. Org. Chem., 61, 8186, 1996) linked oligonucleotides a-f (* = MMI linkage; 5'-GCGT*TT*TT*TT*TT*TGCG-3') containing various combinations of 2'-O-methyl and 2'-fluoro substituent were synthesized as a model to study the global conformational change upon hybridization to the complement RNA. Fourier transform infrared (FTIR) spectroscopic technique has been used to study and compare the influence of these modifications on the solution conformation of 2'-modified MMI DNA-RNA duplexes. FTIR analysis of the single-stranded RNA (5'-CGCAAAAAAAAAACGC-3') and the modified oligonucleotides a-f showed that all sugar residues adopted a C3'-endo conformation (North-type). Stable duplexes were formed when oligonucleotides a-f were hybridized to the complement RNA. These duplexes retained the original C3'-endo conformation for all sugar residues, hallmark of an A-form of duplex. We postulate that the observed preorganization of the sugar residues and oligonucleotides containing 2'-modified MMI modifications may play an important role in both improving the recognition of RNA target and enhancing the stability of duplex formation with RNA.

Carbohydrates↗
PubMed2006

Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration.

4-Hydroxynonenal (4-HNE) is a reactive aldehyde species generated endogenously from the nonenzymatic oxidation of n-6 polyunsaturated fatty acids under physiological conditions. We have reported that intense white light exposure increases 4-HNE-protein modification in the retina prior to the onset of photoreceptor cell apoptosis. To understand the molecular mechanism(s) underlying the retinal degeneration induced by photooxidative stress, we identified 4-HNE-modified retinal proteins using a proteomic approach. Albino rats were exposed to 5 k lx white fluorescent light for 3 h and retinas were removed 24 h later and pooled. By Western dot blot analysis, the total intensity of 4-HNE-modified proteins was increased 1.5-fold following the exposure compared to dim light controls. In two independent sets of two-dimensional gel electrophoresis/Western blots followed by peptide mass fingerprinting (PMF), nine proteins including voltage-dependent anion channel, enolase 1alpha, aldolase C, crystallins alphaA and betaB3, heterogeneous nuclear ribonucleoprotein A2/B1, albumin, and glutamine synthetase were identified. We observed that 4-HNE modifications of retinal proteins are specific to a particular set of proteins rather than random events on abundant proteins. By immunohistochemistry, localization of 3 identified proteins overlapped with immunoreactivity of 4-HNE-modified proteins in light-exposed retinas. Intense light exposure increases 4-HNE-protein modifications on specific retinal proteins in several functional categories including energy metabolism, glycolysis, chaperone, phototransduction, and RNA processing. Together with previous reports that 4-HNE modification changes protein activities, these results suggest a close association of 4-HNE-protein modifications with the initiation of light-induced retinal degeneration.

Aldehydes↗