Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA modifications”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,387 records · Page 77Linked to original sources
PubMed1999

Generation and analysis of GluR5(Q636R) kainate receptor mutant mice.

The physiological significance of RNA editing of transcripts that code for kainate-preferring glutamate receptor subunits is unknown, despite the fact that the functional consequences of this molecular modification have been well characterized in cloned receptor subunits. RNA editing of the codon that encodes the glutamine/arginine (Q/R) site in the second membrane domain (MD2) of glutamate receptor 5 (GluR5) and GluR6 kainate receptor subunits produces receptors with reduced calcium permeabilities and single-channel conductances. Approximately 50% of the GluR5 subunit transcripts from adult rat brain are edited at the Q/R site in MD2. To address the role of glutamate receptor mRNA editing in the brain, we have made two strains of mice with mutations at amino acid 636, the Q/R-editing site in GluR5, using embryonic stem cell-mediated transgenesis. GluR5(RloxP/RloxP) mice encode an arginine at the Q/R site of the GluR5 subunit, whereas GluR5(wt(loxP)/wt(loxP)) mice encode a glutamine at this site, similar to wild-type mice. Mutant animals do not exhibit developmental abnormalities, nor do they show deficits in the behavioral paradigms tested in this study. Kainate receptor current densities were reduced by a factor of six in acutely isolated sensory neurons of dorsal root ganglia from GluR5(RloxP/RloxP) mice compared with neurons from wild-type mice. However, the editing mutant mice did not exhibit altered responses to thermal and chemical pain stimuli. Our investigations with the GluR5-editing mutant mice have therefore defined a set of physiological processes in which editing of the GluR5 subunit is unlikely to play an important role.

Animals↗
PubMed1994

Transient mitochondrial transcript level decay in oxidative stressed chondrocytes.

Steady-state levels of 12S rRNA and NADH dehydrogenase subunit 4 mRNA (ND4) mitochondrial transcripts were measured on rabbit articular chondrocyte in culture. In pseudosynchronized chondrocytes, changes of mitochondrial RNA levels were observed during the progression of the cells in the cell cycle. Oxidative stress generated by the hypoxanthine-xanthine oxidase system (HX-XO) induced a transient decrease in the levels of both ND4 and 12S rRNA. Mitochondrial RNA levels recovered 24 h after the oxidative stress. These RNA level changes were not associated with modifications in the structure or the copy number of the mitochondrial genome. Furthermore, the decrease in the amount of the mitochondrial transcripts observed may be related to a transient inhibition of mitochondrial transcription since the treatment of cells with ethidium bromide (a mitochondrial transcription inhibitor) resulted in the same decrease in 12S rRNA level as HX-XO treatment alone.

Animals↗
PubMed2006

Evolution of small nucleolar RNAs in nematodes.

In contrast to mRNAs, which are templates for translating proteins, non-protein coding (npc) RNAs (also known as 'non-coding' RNA, ncRNA), exhibit various functions in different compartments and developmental stages of the cell. Small nucleolar RNAs (snoRNAs), one of the largest classes of npcRNAs, guide post-transcriptional modifications of other RNAs that are crucial for appropriate RNA folding as well as for RNA-RNA and RNA-protein interactions. Although snoRNA genes comprise a significant fraction of the eutherian genome, identifying and characterizing large numbers of them is not sufficiently accessible by classical computer searches alone. Furthermore, most previous investigations of snoRNAs yielded only limited indications of their evolution. Using data obtained by a combination of high-throughput cDNA library screening and computational search strategies based on a modified DNAMAN program, we characterized 151 npcRNAs, and in particular 121 snoRNAs, from Caenorhabditis elegans and extensively compared them with those in the related, Caenorhabditis briggsae. Detailed comparisons of paralog snoRNAs in the two nematodes revealed, in addition to trans-duplication, a novel, cis-duplication distribution strategy with insertions near to the original loci. Some snoRNAs coevolved with their modification target sites, demonstrating the close interaction of complementary regions. Some target sites modified by snoRNAs were changed, added or lost, documenting a high degree of evolutionary plasticity of npcRNAs.

Animals↗
PubMed1993

Translation initiation complex formation with 30 S ribosomal particles mutated at conserved positions in the 3'-minor domain of 16 S RNA.

Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants. Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA(fMet) and tRNA(Phe) for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA(fMet). The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m2(6)A, in the synthetic RNA. Synthetic particles containing the mutation U1512C, which converts the universal U.G pair to C.G enhanced both tRNA(fMet) binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect. Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3'-minor domain, also enhanced tRNA(fMet) selectivity, in this case by reducing complex formation with elongator tRNA. Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels. The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence. This result had not been detected by standard tRNA-30 S binding assays. Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA(fMet).

Base Sequence↗
PubMed2000

Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA.

Hypusine is formed through a spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine residue. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria known to contain hypusine. Although both eIF-5A and deoxyhypusine synthase are essential genes for cell survival and proliferation, the precise biological function of eIF-5A is unclear. We have previously proposed that eIF-5A may function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., Yan, Y. P., and Chen, K. Y. (1997) Biol. Signals 6, 166-174). Here we used the method of systematic evolution of ligands by exponential enrichment (SELEX) to identify the sequence specificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained after 16 rounds of selection exhibited a significant increase in binding affinity for eIF-5A with an apparent dissociation constant of 1 x 10(-7) m. The hypusine residue was found to be critical for this sequence-specific binding. The post-SELEX RNAs shared a high sequence homology characterized by two conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was determined as AAAUGUCACAC by sequence alignment and binding studies. BLAST analysis indicated that this sequence was present in > 400 human expressed sequence tag sequences. The C terminus of eIF-5A contains a cold shock domain-like structure, similar to that present in cold shock protein A (CspA). However, unlike CspA, the binding of eIF-5A to either the post-SELEX RNA or the 5'-untranslated region of CspA mRNA did not affect the sensitivity of these RNAs to ribonucleases. These data suggest that the physiological significance of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.

Base Sequence↗
PubMed2003

Synthesis, thermal stability and resistance to enzymatic hydrolysis of the oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridines.

The synthesis of oligonucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridine (D) is described, and thermal stability and resistance to enzymatic hydrolysis of the ODNs are compared with ODNs containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine (H). The ODNs containing D and the complementary RNA demonstrated a duplex thermal stabilization of 0.4-3.9 degrees C per modification depending on the position and the number, while the ODNs containing H with the RNA showed slightly less effective thermal stabilization. Further more, the ODNs containing D were found to be more resistant to nucleolytic hydrolysis, not only by snake venom phosphodiesterase (SVPD; a 3'-exonuclease) but also by DNase I (an endonuclease). The half-life of the 17mer containing five molecules of D against nucleolytic hydrolysis by SVPD was 240 times greater than the unmodified 17mer ODN, which is 1.8 times greater than the ODN containing 5Hs in the same sequence. Against DNase I, the same ODN containing 5Ds was 24 times greater stable than the unmodified 17mer and 15 times more stable than the ODN containing 5Hs. We also examined whether the duplexes formed by the ODNs containing D and the complementary RNAs could be a substrate of Escherichia coli RNase H. It was revealed that a minimum of five contiguous unmodified 2'-deoxyribonucleosides between Ds was required to constitute a substrate of E.coli RNase H. Thus, the ODN with Ds and at least five contiguous unmodified 2'-deoxyribonucleosides between Ds was found to be a candidate for a novel antisense molecule.

Base Sequence↗
PubMed1992

Peroxidase oxidizes N-nitrosomethylaniline to ultimate carcinogens(s) binding to DNA and transfer RNA in vitro.

Carcinogenic N-nitrosomethylaniline is oxidized in vitro by horseradish peroxidase in the presence of H2O2 to ultimate carcinogens, which bind to DNA and transfer RNA (tRNA). tRNA is more accessible for modification by the activated carcinogen studied. The modification of nucleic acid by N-nitrosomethylaniline metabolite(s) formed by peroxidase is inhibited by some compounds of physiological importance (ascorbate, glutathine) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-postlabeling assay of DNA and tRNA modified by N-nitrosomethylaniline activated by peroxidase shows covalent adduct formation with nucleic acids. The role of peroxidases in the activation of N-nitrosamines leading to organ and/or cell specificity of these carcinogens is discussed.

Autoradiography↗
PubMed2003

Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatin.

Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states.

DNA Primers↗
PubMed2003

Ribosome structure and activity are altered in cells lacking snoRNPs that form pseudouridines in the peptidyl transferase center.

One of the oldest questions in RNA science is the role of nucleotide modification. Here, the importance of pseudouridine formation (Psi) in the peptidyl transferase center of rRNA was examined by depleting yeast cells of 1-5 snoRNAs that guide a total of six Psi modifications. Translation was impaired substantially with loss of a conserved Psi in the A site of tRNA binding. Depletion of other Psis had subtle or no apparent effect on activity; however, synergistic effects were observed in some combinations. Pseudouridines are proposed to enhance ribosome activity by altering rRNA folding and interactions, with some Psis having greater effects than others. The possibility that modifying snoRNPs might affect ribosome structure in other ways is also discussed.

Base Sequence↗
PubMed1987

Depurination of yeast 26S ribosomal RNA by recombinant ricin A chain.

The nature of the modification of yeast ribosomes by the recombinant form of the ricin A chain has been examined. Evidence is presented that the 26S rRNA molecule is depurinated at a specific site and that the activity is inhibited by antibody raised to ricin A chain. It thus appears that the recombinant form of this toxin retains the depurination activity of the native molecule. These results are consistent with the model that the site of depurination is in a highly conserved sequence forming a loop on the surface of the ribosome, a domain involved in elongation factor-dependent binding of aminoacyl-tRNA.

Purines↗
PubMed1994

Role of glycosylation in the biosynthesis and activity of rabbit testicular angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is a type I glycoprotein anchored in the plasma membrane by a hydrophobic domain near its carboxyl terminus. The enzymatically active extracellular domain of ACE is slowly released from the cell by cleavage-removal of its membrane-anchoring carboxyl-terminal region. In the present study, we investigated the role of N- and O-glycosylation in intracellular transport and extracellular cleavage-secretion of rabbit testicular ACE. For ACE expression, we used an in vitro translation system, a permanently transfected mouse cell line, and human and Chinese hamster cells transiently transfected with vaccinia virus-T7 RNA polymerase-driven expression vectors. Sugar modifications of ACE were analyzed by testing its sensitivity to specific glycosidases. Cellular protein glycosylation was inhibited by using chemical inhibitors and a mutant cell line defective in protein glycosylation. Our experiments demonstrated that newly synthesized ACE acquires both N- and O-linked sugars before its cleavage-secretion and complete blockage of glycosylation results in rapid intracellular turnover of underglycosylated ACE. However, ACE synthesized without N-linked complex sugars and O-linked sugars can undergo normal transport and cleavage-secretion, and the underglycosylated protein is enzymatically active.

Animals↗
PubMed2001

Defective satellite cells in congenital myotonic dystrophy.

In this study we have developed an in vitro cell culture system which displays the majority of the defects previously described for congenital myotonic dystrophy (CDM) muscle in vivo. Human satellite cells were isolated from the quadriceps muscles of three CDM fetuses with different clinical severity. By Southern blot analysis all three cultures were found to have approximately 2300 CTG repeats. This CTG expansion was found to progressively increase in size during the proliferative life span, confirming an instability of this triplet in skeletal muscle cells. The CDM myoblasts and myotubes also showed abnormal retention of mutant RNA in nuclear foci, as well as modifications in their myogenic program. The proliferative capacity of the CDM myoblasts was reduced and a delay in fusion, differentiation and maturation was observed in the CDM cultures compared with unaffected myoblast cultures. The clinical severity and delayed maturation observed in the CDM fetuses were closely reflected by the phenotypic modifications observed in vitro. Since the culture conditions were the same, this suggests that the defects we have described are intrinsic to the program expressed by the myoblasts in the absence of any trophic factors. Altogether, our results demonstrate that satellite cells are defective in CDM and are probably implicated in the delay in maturation and muscle atrophy that has been described previously in CDM fetuses.

Biopsy↗
PubMed1997

Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit.

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.

Fruit↗
PubMed1998

Identification of the pro-oncogene stathmin/op18 mRNA in the brain of mitochondrial Mn-superoxide dismutase-deficient mice by a modified differential display PCR.

Differential gene expression plays a critical role in many biological processes. To facilitate the screening of the entire mRNA species for cloning differentially expressed genes, we have made an effort to merge two polymerase chain reaction (PCR)-based methods, differential display (DD) and arbitrarily primed RNA fingerprinting (APR-FP), with some modifications. Using this modified method to screen the mRNAs of the brain tissues of manganese superoxide dismutase (MnSOD)-deficient mice, we found several differentially expressed mRNA species. One mRNA species that was further analyzed by Northern hybridization and sequencing, and was confirmed to be induced only in the brain of MnSOD-deficient mice, encoded stathmin/op18. The MnSOD deficiency causes oxidative stress and mitochondrial dysfunction. Thus, the induction of stathmin/op18, a gene linked with microtubule catastrophe (disassembly) and often upregulated in neoplastic tissues and proliferating cells, may provide some understanding of the pathological changes in the brain of MnSOD-deficient mice.

Animals↗
PubMed1987

Involvement of lysine and arginine residues in the binding of yeast ribosomal protein YL3 to 5S RNA.

The contribution of lysine and arginine residues to the formation of yeast ribonucleoprotein complex 5S RNA. protein YL3 has been investigated by determining the effects on complex formation of modification with chemical reagents specific for either lysine or arginine. Treatment of protein YL3 with acetic anhydride, maleic anhydride or phenylglyoxal is accompanied by loss of its capacity to bind to 5S RNA. This effect is accomplished by modification with phenylglyoxal of only 3 arginine residues per YL3 molecule. In contrast, a large number of protein YL3 amino groups must be modified by acetic anhydride to prevent complex formation.

Acetic Anhydrides↗
PubMed2006

Chromatin remodelling and transcription: be-WICHed by nuclear myosin 1.

Transcription in eukaryotic cells requires dynamic changes of chromatin structure to facilitate or prevent RNA polymerase access to active genes. These structural modifications rely on the concerted action of ATP-dependent chromatin-remodelling complexes and histone-modifying enzymes, which generate a chromatin configuration that is either compatible with transcription (euchromatin) or incompatible (heterochromatin). Insights into how these structural changes might be coordinated for RNA polymerase I (pol I) genes come from the discoveries of the nucleolar-remodelling complex (NoRC) and B-WICH--a high molecular weight fraction of the WSTF/SNF2h chromatin-remodelling complex. NoRC produces a repressive chromatin state; B-WICH, together with nuclear myosin 1, activates pol I transcription directly on chromatin templates and might also function in the maintenance of ribosomal chromatin structure.

Carrier Proteins↗
PubMed2001

Novel RNA-binding properties of Pop3p support a role for eukaryotic RNase P protein subunits in substrate recognition.

Ribonuclease P (RNase P) catalyzes the 5'-end maturation of transfer RNA molecules. Recent evidence suggests that the eukaryotic protein subunits may provide substrate-binding functions (True, H. L., and Celander, D. W. (1998) J. Biol. Chem. 273, 7193-7196). We now report that Pop3p, an essential protein subunit of the holoenzyme in Saccharomyces cerevisiae, displays novel RNA-binding properties. A recombinant form of Pop3p (H6Pop3p) displays a 3-fold greater affinity for binding pre-tRNA substrates relative to tRNA products. The recognition sequence for the H6Pop3p-substrate interaction in vitro was mapped to a 39-nucleotide long sequence that extends from position -21 to +18 surrounding the natural processing site in pre-tRNA substrates. H6Pop3p binds a variety of RNA molecules with high affinity (K(d) = 16-25 nm) and displays a preference for single-stranded RNAs. Removal or modification of basic C-terminal residues attenuates the RNA-binding properties displayed by the protein specifically for a pre-tRNA substrate. These studies support the model that eukaryotic RNase P proteins bind simultaneously to the RNA subunit and RNA substrate.

Binding Sites↗
PubMed1975

Inhibition of methylated nucleoside synthesis in vivo: accumulation of incompletely methylated transfer RNA in ethionine-treated cells of Escherichia coli B.

tRNA prepared from cells of E. coli B that had been incubated with 0.5% DL-ethionine (Ethio sRNA) was found to accept methyl groups from 14CH3-S-adenosyl-methionine in the enzymatic reaction catalyzed in vitro by tRNA methyl transferases from untreated cells of the same organism. tRNA from cells that were not exposed to ethionine did not accept a significant level of methyl groups when incubated with the same enzyme system. Base ratio analysis of the product obtained after in vitro addition of methyl groups to Ethio sRNA by enzymes from normal E. coli B indicated that a high proportion of uracil sites in this tRNA were available for enzymatic methylation. These results indicated that tRNA from ethionine-treated organisms was recognized by the homologous enzymes to be incompletely methylated, while, as previously shown, all methyl-acceptor sites on tRNA from normal cells were already filled, and that Ethio sRNA was preferentially deficient in methyl groups on uracil moieties in the RNA molecules. Ethionine thus appears to interfere with normal tRNA modification in vivo.

Cell Division↗