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At least 883 records · Page 49Linked to original sources
PubMed1983

Chemical accessibility of the 4.5S RNA in spinach chloroplast ribosomes.

We have examined the accessibility to diethylpyrocarbonate of spinach chloroplast 4.5S ribosomal RNA when free and when it is part of the ribosomal structure. The modifications in free 4.5S RNA were found mostly in single-stranded regions of the secondary structure model proposed in our previous paper (Kumagai, I. et al. (1982) J.B.C. 257, 12924-28): adenines at positions 17, 19, 33, 36, 54, 55, 60, 64, 68, 72, 77, 86 and 87 were identified as the reactive residues. On the other hand, in 4.5S RNA in 70S ribosomes or 50S subunits, adenine 33 was exclusively modified, and its reactivity was much higher than in free 4.5S RNA. This highly accessible A33 of spinach 4.5S RNA is located within a characteristic seven nucleotide sequence, which is found in the 4.5S rRNAs from spinach, tobacco and a fern but deleted in 4.5S RNAs from maize and wheat.

Base Sequence↗
PubMed2001

Linking class-switch recombination with somatic hypermutation.

The recent discovery of a molecular link between two apparently different genetic alteration events--class-switch recombination and somatic hypermutation--has led to the idea that the recognition and cleavage of target DNA in these two events might be mediated by similar or identical molecules to those involved in RNA editing. This could mean that the complexity of mammalian genetic information may be enriched by an interplay between RNA editing and DNA modification.

APOBEC-1 Deaminase↗
PubMed2005

SUMO-1 modification alters ADAR1 editing activity.

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.

Adenosine Deaminase↗
PubMed1972

Isolation of phi X174 specific messenger ribonucleic acids in vivo and identification of their 5' terminal nucleotides.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) hybridization in formamide at low temperature was applied to hybridization of PhiX174 replicative form DNA and in vivo PhiX174 specific messenger RNA (mRNA) with some modification. We found that PhiX174 mRNA up to molecular weight 1.2 x 10(6) could be hybridized to and eluted from DNA without apparent breakage of phosphodiester bonds and the 5' terminal guanosine triphosphate and adenosine triphosphate of the RNA. By alkali hydrolysis of the purified in vivo PhiX174 mRNA and subsequent thin-layer chromatography of the digest, we isolated the 5' terminal nucleotides and identified them as 2'- or 3'-monophosphate guanosine 5'-triphosphate (pppGp) and 2'- or 3'-monophosphate adenosine 5'-triphosphate (pppAp). By comparing the in vitro and in vivo synthesized PhiX174 mRNA, a difference in the pppAp-pppGp ratio was observed. In the in vitro RNA, this ratio was 1.5, whereas in the in vivo RNA it was 5.5.

Adenosine Monophosphate↗
PubMed2004

A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications.

We have modified the yeast two-hybrid system to enable the detection of protein-protein interactions that require a specific post-translational modification, using the acetylation of histones and the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II as test modifications. In this tethered catalysis assay, constitutive modification of the protein to be screened for interactions is achieved by fusing it to its cognate modifying enzyme, with the physical linkage resulting in efficient catalysis. This catalysis maintains substrate modification even in the presence of antagonizing enzyme activities. A catalytically inactive mutant of the enzyme is fused to the substrate as a control such that the modification does not occur; this construct enables the rapid identification of modification-independent interactions. We identified proteins with links to chromatin functions that interact with acetylated histones, and proteins that participate in RNA polymerase II functions and in CTD phosphorylation regulation that interact preferentially with the phosphorylated CTD.

Acetylation↗
PubMed2004

Immunogenicity and protection efficacy of replication-deficient influenza A viruses with altered NS1 genes.

We explored the immunogenic properties of influenza A viruses with altered NS1 genes (NS1 mutant viruses). NS1 mutant viruses expressing NS1 proteins with an impaired RNA-binding function or insertion of a longer foreign sequence did not replicate in murine lungs but still were capable of inducing a Th1-type immune response resulting in significant titers of virus-specific serum and mucosal immunoglobulin G2 (IgG2) and IgA, but with lower titers of IgG1. In contrast, replicating viruses elicited high titers of serum and mucosal IgG1 but less serum IgA. Replication-deficient NS1 mutant viruses induced a rapid local release of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Moreover, these viruses also elicited markedly higher levels of IFN-alpha/beta in serum than the wild-type virus. Comparable numbers of virus-specific primary CD8(+) T cells were determined in all of the groups of immunized mice. The most rapid onset of the recall CD8(+)-T-cell response upon the wild-type virus challenge was detected in mice primed with NS1 mutant viruses eliciting high levels of cytokines. It is noteworthy that there was one NS1 mutant virus encoding NS1 protein with a deletion of 40 amino acids predominantly in the RNA-binding domain that induced the highest levels of IFN-alpha/beta, IL-6 and IL-1beta after infection. Mice that were immunized with this virus were completely protected from the challenge infection. These findings indicate that a targeted modification of the RNA-binding domain of the NS1 protein is a valuable technique to generate replication-deficient, but immunogenic influenza virus vaccines.

Animals↗
PubMed2000

A novel method for the preparation of large cDNA fragments from dengue-3 RNA genome by long RT-PCR amplification.

For many years, dengue viruses were among the most difficult flaviviruses to isolate and to identify, but technical advances in the past 20 years have facilitated this process. Dengue viruses are usually recovered from specimens by the infection of mosquito-cell cultures. The virus may be passaged several times in cell cultures until a sufficient infectivity titer is attained. The viral nucleocapsid consists of capsid protein and an RNA genome. The dengue genome is a single stranded messenger (positive) sense RNA of approximately 11 kb in length. The isolation of dengue genomic RNA from various sources requires precautions to avoid RNases. RNases are released during cell disruption, and their activity must be inhibited as quickly as possible by using guanidinium thiocyanate in the presence of 2-mercaptoethanol. There has recently been a revolution in molecular biology with the development of the powerful reverse transcriptase (RT) and polymerase chain reaction (PCR) technology. Advanced studies on RT technique lead to much further improvement of the reverse transcriptase enzyme by genetic engineering. The Superscript II RNase H- RT (GIBCO BRL, USA) is genetically engineered DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA. DNA or an RNA-DNA hybrid. This enzyme is produced from a cloned M-MLV RT gene constructed by the introduction of point mutation in the RNase H active center. The selective mutations within the RNase H domain maintain full polymerase activity. This structural modification eliminates degradation of RNA molecules during the first strand cDNA synthesis. The combination of thermostable DNA polymerase with and without proofreading activity (3'-exonuclease activity), improved buffer conditions and thermal cycling profiles overcome the length limitation of PCR. On the basis of these findings, we have developed a long RT-PCR system for preparing large cDNA fragments of dengue 3 virus (H-87) by using the Superscript II RNase H- RT for reverse transcription and a mixture of Taq and Pwo DNA polymerases for PCR. Three large cDNA fragments covered the full genomic RNA from the 5'-end to the 3'-end of dengue-3 virus (H-87; 10,696 bps) could be successfully prepared as the lengths of 2.437 bps, 3,980 bps and 4,337 bps respectively. The ability of our developed long RT-PCR will bring speed and simplicity to genomic mapping and sequencing and facilitate studies in molecular genetics of dengue viruses.

Aedes↗
PubMedDate not supplied

Monitoring of RNA clearance in a novel plasmid DNA purification process.

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.

Biochemistry↗
PubMed1991

Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor.

The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield.

Blotting, Northern↗
PubMed1994

Fingerprinting the folding of a group I precursor RNA.

Evidence that folding of the Tetrahymena pre-rRNA follows a defined path and is rate-determining for splicing at physiological temperatures is presented. Structural isomers were separated by native polyacrylamide gel electrophoresis and their splicing activities were compared. GTP binding selectively shifts the active form of the pre-RNA to an electrophoretic band containing both spliced and unspliced RNA. In situ chemical modification provides evidence for base-pair rearrangements in the 5' exon and structural alterations in the intron core of partially and fully active forms. Transition to the fully active precursor requires high temperature, but the activation energy is lower than expected for opening of RNA helices. Implications for control of RNA conformation during splicing are discussed.

Base Sequence↗
PubMed2006

Universal initiator nucleotides for the enzymatic synthesis of 5'-amino- and 5'-thiol-modified RNA.

We report the chemical synthesis of 5'-amino- and 5'-thiol-hexaethylene glycol guanosine nucleotides and their enzymatic incorporation into RNA, followed by chemical modifications at their nucleophilic ends. By using two similar routes, the conjugates of guanosine-5'-monophosphate and hexaethylene glycol with attached reactive groups (SH or NH(2)) were synthesized using phosphoramidite chemistry, and characterized by MALDI TOF mass spectrometry. These initiator molecules were efficiently incorporated into RNA at the 5'-end by run-off transcription using T7 RNA polymerase. The potential of these RNA conjugates for a broad reaction range with electrophiles is shown here, thereby enabling their use for diverse biochemical applications.

Codon, Initiator↗
PubMed1994

A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.

Amino Acid Sequence↗
PubMed2004

Crystallization and preliminary diffraction analysis of a group I ribozyme from bacteriophage Twort.

Group I introns are catalytic RNAs that are capable of performing a variety of phosphotransesterification reactions including self-splicing and RNA cleavage. The reactions are efficient, accurate and dependent only on the presence of guanosine-nucleotide substrate and sufficient magnesium ion to stabilize the structure of the RNA. To understand how the group I intron active-site facilitates catalysis, crystals of a 242-nucleotide ribozyme bound to a four-nucleotide product RNA have been produced that diffract to 3.6 A resolution. The space group of these crystals is I2(1)2(1)2(1) and the unit-cell parameters are a = 94.6, b = 141.0, c = 210.9 A. A single heavy-atom derivative has been synthesized by covalent modification of the product RNA with iodine.

Base Sequence↗
PubMed2004

Phosphorylation of BRCA2 by the Polo-like kinase Plk1 is regulated by DNA damage and mitotic progression.

The breast cancer susceptibility protein, BRCA2, preserves chromosomal stability through roles in the repair of DNA double-strand breaks, and possibly, cell division. Post-translational modifications that may coordinate these functions remain poorly characterized. Here, we report that BRCA2 is a substrate for the mitotic Polo-like kinase, Plk1. BRCA2 undergoes phosphorylation in cells synchronously passing through the G2/M phases of cell cycle, when Plk1 expression and activity are maximal. Depletion of Plk1 by RNA interference suppresses BRCA2 modification. BRCA2 and Plk1 interact with one another in cell lysates, through a conserved region in BRCA2, which spans the eight BRC repeat motifs essential for its function in DNA repair. Within this region, residues positioned between BRC repeats--but not the repeat motifs themselves--are phosphorylated by Plk1. Interestingly, Plk1-mediated modification of BRCA2 during the G2/M phases is inhibited by treatment with the radiomimetic agent, adriamycin. Thus, our findings define a regulatory circuit for BRCA2 phosphorylation by Plk1 that is responsive to DNA damage as well as mitotic progression.

BRCA2 Protein↗
PubMedDate not supplied

[Biological consequences of the decay of 3H and 14C incorporated into the influenza virus].

An influenza virus labeled with 3H-uridine loses its infectiousness when stored for a long time. It is suggested that disintegration of tritium incorporated into virus RNA causes lethal intramolecular modifications therein. At the same time, the antigenic activity of virus nucleoprotein decreases perhaps due to the direct effect of tritium. The comparison of the degree of inactivation of various antigenic sites of the nucleoprotein within a virus labeled with 3H-uridine, suggests that they are located at different distances from RNA. A long-term action of 3H disintegration on RNA of a maturing virus decreases the yield probably due to the injury of the intracellular virus RNA during the infections process. Upon storage of the influenza virus labelled with 14C-amino acids the antigenic properties are reduced by the nucleoprotein while the infectiousness remains unaffected. The long-term effect of 14C disintegration on proteins of the maturing virus does not lead to fatal outcome.

Antigens, Viral↗
PubMed1980

Post-transcriptional regulation of gene expression in guinea pig tissues.

The formation of individual functional mRNA sequences in higher organisms requires many steps in addition to transcription. These include RNA splicing, polyadenylation, base modification, transport from nucleus to cytoplasm and assembly into polyribosomes. Various control mechanisms must also operate. These will function on a quantitative basis to account for the differing frequency of the various classes of cytoplasmic mRNAs, and also on a qualitative basis, because in higher organisms not all the nuclear poly(A)-containing RNA molecules are found in a cytoplasmic poly(A)-containing RNA population from the same tissue. During our studies on the mechanisms controlling the accumulation of the poly(A)-containing RNA sequences which occur with high and moderately high frequency in the cytoplasm of the lactating guinea pig mammary gland, it became apparent that > 75% of the 20,000 or so poly(A)-containing nuclear RNA sequences were not found in the cytoplasmic poly(A)-containing RNA fraction. Here we demonstrate that many of the poly(A)-containing RNA sequences retained in the nucleus of the lactating guinea pig mammary gland are also present in the nucleus and cytoplasm of the liver of the male guinea pig. These observations provide new evidence for a predominant role of post-transcriptional mechanisms in the regulation of structural gene expression in guinea pig tissue.

Animals↗
PubMed1975

Structural and transcriptional features of the mouse spermatid genome.

A whole-mount electron microscope technique has allowed direct visualization of the transcription process in mouse spermatids. Thes observations have been supported by light and electron microscope autoradiographic techniques that employ [3H]uridine and [3H]arginine in attempts to clarify mechanisms of RNA synthesis and their relationship to nuclear histone changes throughout spermiogenesis. Early spermatid genomes are dispersed almost completely, whereas in later spermiogenic steps the posterior or flagellar nuclear region is readily dispersed and the anterior or subacrosomal nuclear region remains compact. Display of genome segments permits identification of regions where transcription complexes, presumably heterogeneous nuclear RNA species, are seen related to chromatin. These complexes appear as ribonucleoprotein chains, some of them of considerable length, decreasing progressively in number in late spermiogenic steps. This decrease coincides with diminishing rates of [3H]uridine incorporation. Two distinct patterns of chromatin have been identified: a beaded chromatin type associated with transcription complexes encounterd in early spermatids; and a smooth chromatin type not involved in transcriptive activity observed in advanced spermiogenic genomes. Protein particles staining densely with phosphotungstic acid become apparent in nuclei of spermatids after [3H]arginine incorporation becomes significant. There is no structural or autoradiographic evidence for the presence of nucleoli during spermiogenesis. From these data and from previous experimental findings, we conclude that: (a) spermatogonia, spermatocytes and Sertoli cells are transcriptionally expressed into heterogeneous nuclear RNA and preribosomal RNA species whereas transcription in spermatids is predominantly heterogeneous nuclear RNA; and (b) the modification of the chromatin patterns in late spermiogenic steps indicates a stabilized genome that restricts transcriptive functions.

Animals↗