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At least 1,459 records · Page 81Linked to original sources
PubMed1989

Inhibition of radiation induced changes in testicular RNA content by a thiophosphate.

Radiation induced changes in the testicular RNA content of whole body irradiated Swiss albino mouse and their modification by a thiophosphate, WR-2721 were studied. Three different doses of gamma radiation were given for the purpose. The RNA content of the testis was found to be affected adversely after irradiation. It was observed that the drug inhibited the alterations in the RNA content to a significant extent. Degree of protection and possible mechanisms of action have been discussed.

Amifostine↗
PubMed2001

Quantification of in vitro retroviral replication using a one-tube real-time RT-PCR system incorporating direct RNA preparation.

The methodological and logistic benefits gained from assessing in vitro antiretroviral replication using one-tube real-time RT-PCR procedures are currently diminished by a continuing need for prior RNA isolation. We now report a simple and inexpensive modification of a commercially available one-tube RT-PCR assay, consisting of detergent-based virus lysis in the presence of a ribonuclease inhibitor, which can be used to directly quantify retroviral RNA levels in culture supernatant. This approach circumvents the potential loss of RNA inherent to RNA-isolation procedures based on prior extraction and demonstrates a dynamic range of at least 4 logs. Using in vitro culture systems incorporating either HIV-1 or FIV, we show that this ability to isolate retroviral RNA directly during the RT-PCR process can provide an equivalent alternative to one of the more time and resource-consuming steps in quantifying in vitro retroviral RNA levels.

Animals↗
PubMed2025

A survey of dietary effects on tRNA abundance and modifications.

Transfer RNAs (tRNAs) play a central role in protein translation and are increasingly recognized as dynamic regulators of gene expression. Both physiological and environmental signals can modulate tRNA abundance and chemical modifications, yet the impact of dietary cues on the tRNA landscape remains poorly understood. Here, we investigated the effects of two distinct dietary interventions-low-protein and high-fat diets-on tRNA abundance and modification profiles across multiple mouse tissues. We conducted a comprehensive analysis of tRNA abundance and modification changes in response to these nutritional challenges using RNA mass spectrometry and Ordered Two-Template Relay sequencing (OTTR-seq), a modified-base-sensitive tRNA sequencing method. Our results reveal both shared and tissue-specific alterations in abundance and modifications of specific nuclear and mitochondrial genome-encoded tRNAs in response to dietary conditions at isotype, isoacceptor, and isodecoder levels. As many of the tissue-specific or diet-responsive tRNA modifications have been previously reported to affect decoding efficiency or translational fidelity, these results have implications for understanding translational adaptation in response to dietary conditions.

Journal Article↗
PubMed1977

Nucleotide sequence neighbouring a late modified guanylic residue within the 28S ribosomal RNA of several eukaryotic cells.

The nucleotide sequence of a particular T1 oligonucleotide found in 41S and 28S RNAs of several cellular cell lines (human, mouse, rat and chicken fibroblast) but absent in 45S ribosomal RNA has been deduced. Its primary structure : A-U-U*-G*-psi-U-C-A-C-C-C-A-C-U-A-A-U-A-Gp shows the presence of a modified G residue which explains the existence of this oligonucleotide in the T1 fingerprint of 41S RNA and 28S. Its absence on the 45S RNA T1 fingerprint is accounted for by a late modification.

Animals↗
PubMed2003

Epigenetic gene regulation by noncoding RNAs.

Functional noncoding RNAs have distinct roles in epigenetic gene regulation. Large RNAs have been shown to control gene expression from a single locus (Tsix RNA), from chromosomal regions (Air RNA), and from entire chromosomes (roX and Xist RNAs). These RNAs regulate genes in cis; although the Drosophila roX RNAs can also function in trans. The chromatin modifications mediated by these RNAs can increase or decrease gene expression. These results suggest that the primary role of RNA molecules in epigenetic gene regulation is to restrict chromatin modifications to particular regions of the genome. However, given that RNA has been shown to be at the catalytic core of other ribonucleoprotein complexes, it is also possible that RNA also plays a role in modulating changes in chromatin structure.

Animals↗
PubMed1995

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.

Base Sequence↗
PubMed2003

A snoRNA that guides the two most conserved pseudouridine modifications within rRNA confers a growth advantage in yeast.

Ribosomal RNAs contain a number of modified nucleotides. The most abundant nucleotide modifications found within rRNAs fall into two types: 2'-O-ribose methylations and pseudouridylations. In eukaryotes, small nucleolar guide RNAs, the snoRNAs that are the RNA components of the snoRNPs, specify the position of these modifications. The 2'-O-ribose methylations and pseudouridylations are guided by the box C/D and box H/ACA snoRNAs, respectively. The role of these modifications in rRNA remains poorly understood as no clear phenotype has yet been assigned to the absence of specific 2'-O-ribose methylations or pseudouridylations. Only very recently, a slight translation defect and perturbation of polysome profiles was reported in yeast for the absence of the Psi at position 2919 within the LSU rRNA. Here we report the identification and characterization in yeast of a novel intronic H/ACA snoRNA that we called snR191 and that guides pseudouridylation at positions 2258 and 2260 in the LSU rRNA. Most interestingly, these two modified bases are the most conserved pseudouridines from bacteria to human in rRNA. The corresponding human snoRNA is hU19. We show here that, in yeast, the presence of this snoRNA, and hence, most likely, of the conserved pseudouridines it specifies, is not essential for viability but provides a growth advantage to the cell.

Base Sequence↗
PubMed1990

Messenger RNA editing and the genetic code.

Messenger RNA editing is defined as a process leading to predetermined modifications of the coding region of a primary gene transcript. By this definition, splicing processes are special forms of editing; however, they are not dealt with in this review. Editing processes different from splicing have been defined in mammalian cells, in RNA viruses, and in mitochondria of trypanosomes, higher plants and vertebrates. These post- or co-transcriptional processes involve addition, deletion, or modification-substitution of nucleotides, and represent previously unrecognized mechanisms for altering the coding potential of a gene and for modulating gene expression.

Animals↗
PubMed1988

Affinity labeling at the A-site of Escherichia coli ribosomes by a non-hydrolyzable gamma-amide analog of GTP.

gamma-Amides of GTP and affinity and photoaffinity derivatives of gamma-amides of GTP: gamma-anilide of GTP, gamma-(4-azido)anilide of GTP, gamma-[N-(4-azidobenzyl)-N-methyl]amide of GTP, gamma[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP and gamma-[4-N-(2-oxoethyl)-N-methylaminobenzyl]amide of GTP substituted efficiently for GTP in the EF-Tu-dependent transfer of aminoacyl-tRNA to the ribosome but, in contrast to GTP, they were not hydrolyzed in this process. They represent a new class of non-hydrolyzable GTP analogs with preserved gamma-phosphodiester bond. The radioactive analog of GTP: gamma-[4-N-(2-chloroethyl)-N-methylamino[14C]benzyl]amide of GTP was used as an affinity labeling probe for the identification of components of the GTPase center formed in the EF-Tu-dependent transfer reaction of aminoacyl-tRNA to the ribosomal A-site. Within a six-component complex of poly(U)-programmed E. coli ribosomes with elongation factor Tu, Phe-tRNA(Phe) (at the A-site), tRNA(Phe) (at the P-site) and the [14C]GTP analog, mainly the ribosomal 23S RNA and to a lesser extent the ribosomal proteins L17, L21, S16, S21 and the ribosomal 16S RNA were labeled by the reagent. No significant modification of EF-Tu was detected.

Affinity Labels↗
PubMed1999

Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA.

Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA. These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433. Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites.

Base Sequence↗
PubMed1979

Selective inhibition of precursor incorporation into ribosomal RNA in gamma-irradiated Tetrahymena pyriformis.

Sublethal doses of gamma radiation are known to inhibit total RNA synthesis in the ciliate protozoan Tetrahymena. To determine if the synthesis of a particular class of RNA is preferentially inhibited, pulse-labeled RNA was isolated from normal exponentially growing cells, irradiated cells, and cells in which total RNA synthesis had recovered to the pre-irradiation level. The RNAs were analyzed by SDS-polyacrylamide gel electrophoresis and oligo(dT)-cellulose column chromatography. Inhibition of RNA synthesis primarily involves ribosomal RNA. However, radiation does not cause a delay in the processing of precursor rRNA or a preferential loss of either of the mature rRNAs. Following irradiation, poly(A)-containing RNA [poly(A+)RNA] is synthesized at a rate up to three times greater than the control rate. The elevated poly(A+)RNA synthesis occurs during the period of depressed rRNA synthesis and even after rRNA synthesis has recovered to its pre-irradiation rate. While the sizes of the total cellular ribonucleoside triphosphate pools are depressed in the irradiated cells, these pools probably do not represent the actual compartments containing the precursors for RNA synthesis, and the observed changes cannot explain the modifications in macromolecular synthesis in irradiated Tetrahymena.

Gamma Rays↗
PubMed1993

Limitations and modifications of quantitative polymerase chain reaction. Application to measurement of multiple mRNAs present in small amounts of sample RNA.

Quantification of mRNA by polymerase chain reaction (PCR) is performed by using a competitor DNA standard in the PCR or by employing an internal standard RNA following simultaneous reverse transcription (RT) with the sample RNA. The latter approach is more reliable since it accounts for variations in both the RT and PCR steps. However, we describe in this report that at times the internal standard RNA competes with the target mRNA in the PCR when both are present in disproportionate concentrations in the initial simultaneous RT reaction. To overcome the competition in the PCR, multiple simultaneous RT reactions with the sample RTA and the internal standard RNA are required. Such procedures make the approach time consuming and restrict the use of internal standard RNA for quantification of multiple mRNAs present in small amounts of sample RNA. These limitations are circumvented by the competitor DNA standard approach and mRNA levels can be quantified by calculating the RT efficiency. We illustrate the situations by quantifying the levels of IL-2, IL-4, IL-5, IFN-gamma, and beta-actin mRNAs in mitogen stimulated murine T cells using a multiple mRNA specific internal standard.

Animals↗
PubMed2004

Blotting analysis of native IRP1: a novel approach to distinguish the different forms of IRP1 in cells and tissues.

Iron regulatory protein 1 (IRP1) is a bifunctional protein, which either has aconitase activity or binds to specific mRNA structures to regulate the expression of iron proteins. Using recombinant human IRP1, we found that the two functional forms are resolved by nondenaturing polyacrylamide gel electrophoresis and that they are distinguished from IRP1/RNA complexes. This allowed us to use specific antibodies to develop a blotting system that recognized the iron-free and iron-containing IRP1 forms in the soluble fraction and the RNA-bound IRP1 in the high-speed precipitate fraction of cell extracts. The system was used to study IRP1 in HeLa, K562 cells, and monocytes/macrophages before and after treatment with iron salts, iron chelators, or hydrogen peroxide, as well as in stomach and duodenum biopsies. The results showed that iron-bound aconitase IRP1 is by far the prevalent form in most cells and that the major effect of cellular iron modifications is a shift between free and RNA-bound IRP1. The fraction of RNA-bound IRP1 was highly variable among different cells and was often a minor one. Furthermore, blotting showed that electrophoretic mobility shift assay, as commonly used, tends to under-evaluate the amount of total IRP1 and to over-evaluate the actual RNA-binding activity of IRP1. In conclusion, blotting analysis of IRP1 is a new, useful, and convenient method to analyze the amount and conformations of the protein that reveals previously undetected differences in IRP1 compartmentalization among various cell types.

Blotting, Western↗
PubMed1985

Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase.

The structure of the intervening sequence (IVS) of the Tetrahymena rRNA precursor mediates cleavage-ligation reactions that result in pre-rRNA splicing and IVS cyclization. We have developed a method for RNA structure analysis and applied it to the circular form of the IVS RNA. The native RNA was treated with dimethyl sulfate or diethyl pyrocarbonate to modify bases not involved in secondary or tertiary interactions. The RNA was then used as a template for reverse transcription. Elongation of synthetic oligodeoxynucleotide primers was found to stop (or pause) one nucleotide prior to 1-methyladenosine, 3-methylcytidine, and 7-ethoxycarbonyladenosine residues. The detection of 1-methyladenosine is particularly useful for locating single-stranded regions. After chemical cleavage of the RNA, 7-methylguanosine also could be detected. In general, the sites of modification were consistent with a previous model of the secondary structure of the linear form of the IVS RNA, a model based on enzymatic cleavage data, free energy calculations, and phylogenetic comparison. Thus, IVS RNA autocyclization does not involve major rearrangements of the secondary structure, although there is evidence for a conformational change in one region of the molecule. The methods described here should be of general use for obtaining information about structure far from the ends of RNA molecules.

Animals↗
PubMed1980

Post-transcriptional modifications of oat coleoptile ribonucleic acids. 5'-Terminal capping and methylation of internal nucleosides in poly(A)-rich RNA.

Evidence is presented for the occurrence of 5'-terminal capping structures in poly(A)-rich RNAs from oat coleoptile tissue. Similar to the cap structures in mRNA from other eukaryotic organisms, the 5' terminus of these oat coleoptile RNA molecules consists of 7-methylguanosine joined 5' to 5' with the adjacent (penultimate) nucleoside by means of three phosphate groups in two pyrophosphate linkages. The penultimate nucleoside contains primarily purine bases, but small amounts of pyrimidines (cytidine) are also detectable. Some monophosphorylated 5'-termini were also detected, however, they appear to occur as a result of RNA degradation. In addition to the 5 cap, oat RNA molecules are also post-transcriptionally modified with a low frequency of N6-methylations of internal adenosines.

Edible Grain↗
PubMed2002

Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells.

Inactivation of the X chromosome during early female development and the subsequent maintenance of this transcriptionally inert state through countless cell divisions remain a paradigm for epigenetic regulation in mammals. Nevertheless, the exact mechanisms underlying this chromosome-wide silencing process remain unclear. Using differentiating female embryonic stem (ES) cells as a model system, we recently found that histone H3 tail modifications are among the earliest known chromatin changes in the X inactivation process, appearing as soon as Xist RNA accumulates on the X chromosome, but prior to transcriptional silencing of X-linked genes (Heard et al., 2001). In this report we present an integrated analysis of the sequence of early events and chromatin modifications underlying X inactivation in differentiating female ES cells. We have extended our previous analysis concerning changes in histone tail modification states. We find that the hypomethylation of Arg-17 and that of Lys-36 on histone H3 also characterize the inactive X chromosome, and that these profiles show a similarly early onset during the initiation of X inactivation. In addition, we have investigated the kinetics of the shift in replication timing of the X chromosome undergoing inactivation. This event occurs slightly later than Xist RNA coating and the chromatin modifications. Finally, from an early stage in the X inactivation process, characteristic histone modification patterns can be found on the X chromosome at mitosis, suggesting that they represent true epigenetic marks of the inactive state.

Acetylation↗