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PubMedDate not supplied

Human genes for U2 small nuclear RNA map to a major adenovirus 12 modification site on chromosome 17.

U2 RNA is one of the abundant, highly conserved species of small nuclear RNA (snRNA) molecules implicated in RNA processing. As is typical of mammalian snRNAs, human U1 and U2 are each encoded by a multigene family. In the human genome, defective copies of the genes (pseudogenes) far outnumber the authentic genes. The majority or all of the 35 to 100 bona fide U1 genes have at least 20 kilobases (kb) of nearly perfect 5' and 3' flanking homology in common with each other; these U1 genes are clustered loosely in chromosome band 1p36 (refs 5, 7) with intergenic distances exceeding 44 kb. In contrast, the 10 to 20 U2 genes are clustered tightly in a virtually perfect tandem array which has a strict 6-kb repeating unit. We report here the assignment, by in situ hybridization, of the U2 gene cluster to chromosome 17, bands q21-q22. Surprisingly, this region is one of three major adenovirus 12 modification sites which undergo chromosome decondensation ('uncoiling') in permissive human cells infected by highly oncogenic strains of adenovirus. The two other major modification sites, 1p36 and 1q21, coincide with the locations of U1 genes and class I U1 pseudogenes, respectively. We suggest that snRNA genes are the major targets of viral chromosome modification.

Adenoviruses, Human↗
PubMed2002

Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe).

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.

Alkyl and Aryl Transferases↗
PubMed1990

RNA synthesis and processing in the gerbil brain after transient hindbrain ischaemia.

Ribonucleic acid (RNA) synthesis was investigated in gerbils subjected to 15 min transient hindbrain ischaemia using [2-14C]uridine autoradiography. Distribution of synthesized RNA in the subcellular fraction of the tissue was detected by differential centrifugation and density gradient separation using Whittaker's method. In [2-14C]uridine autoradiography, uptake of the tracer into the RNA fraction was not reduced after transient ischaemia. Distributional analysis of [2-14C]uridine in the subcellular fractions revealed that tracer activity in the P3 (microsomes) fraction decreased in the ischaemic regions and tended to decrease in the P4 (ribosomes) fraction, although not significantly. Tracer activity in the P1 (nuclei and cell debris) and P2 (mitochondria, myelin and nerve ending particles) fractions did not decrease. These results indicate that RNA synthesis in the nuclei is not inhibited by ischaemia, but RNA processing is disturbed by the level of the transport. Modification of RNA synthesis and processing by transient ischaemia may influence protein synthesis.

Animals↗
PubMed1982

Effect of incubation and translation inhibitors on the transcriptional activity in salivary glands of Chironomus thummi.

Short preincubations of excised salivary glands of Chironomus thummi in synthetic media modify both the activity of uridine uptake into the cells and its incorporation into RNA. The modification of uptake varies with the medium used. Incorporation into total RNA as well as into nucleolar preribosomal RNA is considerably decreased, while incorporation into non-nucleolar RNAs is little affected. When preincubated explanted glands are briefly treated with the protein synthesis inhibitors cycloheximide or anisomycin, the incorporation activity into preribosomal RNA is slightly recovered. This contrasts with the decrease of the labelling of preribosomal glandular RNA, when those drugs are applied in vivo to the larvae.

Animals↗
PubMed2026

Maternal immune activation perturbs the brain epitranscriptome.

Maternal immune activation (MIA) results in abnormal fetal neurodevelopment and an increased risk of neurodevelopmental disorders. Altered RNA translation has been implicated in the pathophysiology of MIA-associated neurodevelopmental deficits, but more precise mechanisms underlying disruption in RNA metabolism are lacking. Here, we characterize key components of the RNA epitranscriptomic machinery, which refers to the set of reversible chemical modifications on RNA molecules that influence RNA function, including translation, stability, splicing, and localization. Using spatial transcriptomics, we define cell type- and brain region-specific distribution of epitranscriptome regulators in the developing mouse brain. We also use direct RNA sequencing to define how MIA changes the brain epitranscriptome landscape. We identify the demethylase FTO as being notably perturbed in the context of MIA. Using pharmacological and genetic approaches, we target FTO to ameliorate behavioral phenotypes in MIA offspring. In total, this work expands upon mechanisms of translational misregulation in MIA and identifies new targets for therapeutic manipulation.

Animals↗
PubMed1980

[Effect of ethidium bromide on the electrical activity of nerve cells].

The interaction of ethidium bromide with stretch receptor neurons of the crayfish has been studied. The fluorescent staining of the neuron axon membrane was shown to be accompanied with the inactivation of its electrical action potential generation capacity. Taking into account the high specificity of ethidium bromide fluorescence rising under condition of its complexation with nucleic acids especially, the existence of RNA in composition of axon membrane electrogenic channels is suggested. The modification of RNA by ethidium bromide may lead to the inactivation of electrogenic channels in the axon membrane.

Animals↗
PubMed2000

Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.

A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.

DNA, Complementary↗
PubMed1994

Progress towards gene therapy for HIV infection.

The retroviral life cycle and genetic plasticity of human immunodeficiency virus 1 (HIV-1) present unprecedented therapeutic challenges. Twelve years into the HIV epidemic, satisfactory treatment remains elusive. Our current understanding of AIDS pathogenesis calls for early intervention with antiviral agents. Although still in its infancy, human gene therapy holds considerable potential for the long-term treatment of genetic disorders, cancer and chronic infectious diseases. Gene therapy for HIV infection is receiving particularly intensive study: approaches that are in development include both immunotherapy (e.g. therapeutic vaccines and adoptive transfer of CD8+ T-cell clones) and direct antiviral therapy (intracellular immunization). The latter strategies include transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes and modifications of cellular proteins (e.g. intracellular antibodies, soluble CD4). Several of these strategies are now entering clinical trials. While significant conceptual and technical hurdles remain to be overcome before the promise of gene therapy for HIV infection can be fully realized, progress in this field is likely to be rapid and to contribute to the broader applicability of human gene therapy to the treatment of other disorders.

Clinical Protocols↗
PubMed1991

Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle.

We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.

Animals↗
PubMed1990

Transcription elongation and eukaryotic gene regulation.

Each step in the synthesis of functional transcript by RNA polymerase II provides a level at which gene expression can be regulated. Control over the elongation phase of transcription is a recognized regulatory mechanism in prokaryotes; however, only recently have examples of conditional transcription elongation blockage been reported in eukaryotic cellular genes. In several cases, control over transcription elongation clearly contributes to the regulated expression of these genes. Indeed, reports that transcription by RNA polymerase II is initiated and paused on many Drosophila promoters, prior to induction of gene expression, suggests that release of an arrested polymerase, as opposed to polymerase recruitment to a disengaged promoter, may be the key regulatory step for many genes thought to be controlled by transcription initiation (Rougvie & Lis, 1988). RNA polymerase II undergoes modifications, such as association with ancillary elongation factors and phosphorylation of its large subunit carboxy terminal domain (CTD), at stages subsequent to recruitment to a promoter and establishment of a pre-initiation complex (Reinberg & Roeder, 1987; Rappaport et al., 1987; Payne et al., 1989; Laybourn & Dahmus, 1989). It is possible that modifications such as these, or others occurring prior to, during or following transcription initiation, may alter the holoenzyme's transcription elongation properties, to allow recognition or read-through of elongation block signals within a transcription unit. In this review, we will present features of transcription elongation blockage in several eukaryotic cellular genes in the context of our understanding of attenuation and premature transcription termination in prokaryotic and viral genes. We will also present evidence supporting the model that modifications to the RNA polymerase II transcription complex are pivotal to the control of transcriptional at the level of elongation.

Animals↗
PubMed2000

Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii.

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N(6)-methyladenosine-1501, and N(6),N(6)-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N(6)-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.

Animals↗
PubMedDate not supplied

[Affinity modification of DNA-dependent RNA-polymerase of phage T7 with 5'-p-fluorosulfonylbenzoyladenosine].

The affinity modification of the DNA-dependent RNA-polymerase of bacteriophage T7 was carried out by using the specific irreversible inhibitor, 5'-p-fluorosulfonylbenzoyladenosine. The inhibitor was found to bind to the enzyme's active site; the kinetic constants of the modification were calculated. The stoichiometry of the covalent E.I-complex formed was determined by using the 14C-labeled inhibitor.

Adenosine↗
PubMed1985

Initiation of transcription by bacteriophage T4-modified RNA polymerase independently of host sigma factor.

After infection of Escherichia coli with bacteriophage T4 a series of modifications of RNA polymerase takes place including the association of several small polypeptides. We isolated RNA polymerase from cells abortively infected with a series of T4 mutants which arrest phage development at different stages and found that different sets of associated proteins are present in RNA polymerase in each case. The patterns of associated polypeptides seem to correlate with DNA content in the infected cells, suggesting that some of them can be involved both in DNA replication and in the transcription apparatus. One of the modified forms of RNA polymerase contains stoichiometric amounts of a protein with Mr = 25,000 (25K protein), which remains associated with the core enzyme after the removal of sigma factor by chromatography on phosphocellulose. The 25K protein was purified to homogeneity and its effect on transcription selectivity was analyzed in an in vitro system using fragments of T4 DNA as templates. The 25K protein exists in two functional forms which direct core RNA polymerase to utilize two different types of transcription start sites (class I and class II promoters). Both activities do not require host sigma factor. The two forms of 25K protein seem to compete with each other for the core enzyme. The isolated 25K protein can form stable dimers, suggesting that its two activities are associated with the dimeric and monomeric forms. Class I (but not class II) promoters can also be utilized in response to the host sigma factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiography↗
PubMed2006

Medically assisted procreation and transmission of hepatitis C virus: absence of HCV RNA in purified sperm fraction in HIV co-infected patients.

OBJECTIVE: The risk of hepatitis C virus (HCV) transmission in medically assisted procreation (MAP) is debated and some researchers have proposed to exclude MAP for HCV-positive infertile patients. The objectives of this study were to assess the presence of viral RNA in the final preparation of density gradient semen fractions collected from men with chronic HCV and HIV co-infection participating in a MAP program, and to assess whether HIV co-infection influences the rate of the presence of HCV RNA in the semen. DESIGN AND METHODS: The study was based on a cohort of 170 HCV-infected male patients (93 HIV co-infected) participating in a MAP program in a French center. Semen samples were subjected to standard MAP sperm preparation, using density-gradient centrifugation with 40 and 90% layers. All aliquots were tested with a commercially available HCV RNA assay (Roche Monitor), adapted for use with semen after a nucleic HCV RNA extraction modification (Organon Technika). RESULTS: Seminal plasma samples from 19 (11%) patients were HCV RNA positive. The positive HCV viral load in semen was less than 600 IU/ml. None of the 90% fractions from HCV-infected patients were HCV RNA positive. Among the 93 co-infected patients, 10 were positive for HCV RNA in semen and three were HIV/HCV RNA positive in semen. CONCLUSIONS: Although HCV RNA was found in the semen of 11% of patients, no purified sperm fraction, or spermatozoa used in MAP were HCV RNA positive. The 90% purified sperm fraction discards the virus and must be used with care in MAP.

Adult↗
PubMed2006

5'-Triphosphate RNA is the ligand for RIG-I.

The structural basis for the distinction of viral RNA from abundant self RNA in the cytoplasm of virally infected cells is largely unknown. We demonstrated that the 5'-triphosphate end of RNA generated by viral polymerases is responsible for retinoic acid-inducible protein I (RIG-I)-mediated detection of RNA molecules. Detection of 5'-triphosphate RNA is abrogated by capping of the 5'-triphosphate end or by nucleoside modification of RNA, both occurring during posttranscriptional RNA processing in eukaryotes. Genomic RNA prepared from a negative-strand RNA virus and RNA prepared from virus-infected cells (but not from noninfected cells) triggered a potent interferon-alpha response in a phosphatase-sensitive manner. 5'-triphosphate RNA directly binds to RIG-I. Thus, uncapped 5'-triphosphate RNA (now termed 3pRNA) present in viruses known to be recognized by RIG-I, but absent in viruses known to be detected by MDA-5 such as the picornaviruses, serves as the molecular signature for the detection of viral infection by RIG-I.

Animals↗
PubMed1994

The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.

Adenosine Deaminase↗
PubMed2006

Post-transcriptional nucleotide modification and alternative folding of RNA.

Alternative foldings are an inherent property of RNA and a ubiquitous problem in scientific investigations. To a living organism, alternative foldings can be a blessing or a problem, and so nature has found both, ways to harness this property and ways to avoid the drawbacks. A simple and effective method employed by nature to avoid unwanted folding is the modulation of conformation space through post-transcriptional base modification. Modified nucleotides occur in almost all classes of natural RNAs in great chemical diversity. There are about 100 different base modifications known, which may perform a plethora of functions. The presumably most ancient and simple nucleotide modifications, such as methylations and uridine isomerization, are able to perform structural tasks on the most basic level, namely by blocking or reinforcing single base-pairs or even single hydrogen bonds in RNA. In this paper, functional, genomic and structural evidence on cases of folding space alteration by post-transcriptional modifications in native RNA are reviewed.

Base Sequence↗
PubMed2002

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis.

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.

Base Sequence↗