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 1,369 records · Page 76Linked to original sources
PubMed2026

A Unified Mechanism of +1 Ribosomal Frameshifting.

Ribosomes decode 3-nucleotide codons and move in 1-codon increments to maintain the messenger RNA (mRNA) frame thereby accurately producing the encoded protein. In special cases, including viral genomes and regulatory cellular proteins, frameshifting occurs to expand the coding repertoire of an mRNA to make more than one protein. How these frameshifting events are induced and regulated is an active area of research. Here, we discuss recent progress in the understanding of +1 frameshifting (+1FS), during which the ribosome shifts by 1 mRNA nucleotide in the 3' direction. Structural and biochemical studies yielded insights into +1FS induced by mRNA slippery sequences and transfer RNA (tRNA) stem-loop expansion or modifications. tRNAs with an additional anticodon nucleotide are explored as a biotechnology tool for expanding the genetic code in an approach termed quadruplet decoding. We revisit the challenges of the quadruplet decoding model, discuss +1FS scenarios in bacteria and eukaryotes, and propose a unifying structural mechanism for +1FS.

Frameshifting, Ribosomal↗
PubMed2002

Path of an RNA ligand around the surface of the vaccinia VP39 subunit of its cognate VP39-VP55 protein heterodimer.

VP39 is a vaccinia virus-encoded RNA modifying protein with roles in the modification of both mRNA ends. At the 3' end it acts as a processivity factor for the vaccinia poly(A) polymerase (VP55), promoting poly(A) tail elongation. Despite VP39's three-dimensional structure having been elucidated along with details of its mode of mRNA 5' end binding, the VP39-VP55 heterodimer's molecular mechanism of processivity is largely unknown. Here, the area immediately above almost the entire surface of the VP39 subunit was probed using chemical reporters, and the path of a previously unidentified RNA binding site was revealed. The path was indicated to fall within a cleft formed by the intersubunit interface and was consistent with both a previously reported model of the heterodimer-nucleic acid ternary complex and the known function of the heterodimer in processive poly(A) tail elongation.

Binding Sites↗
PubMed1995

Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop.

The structure of the RNA binding target for Saccharomyces cerevisiae ribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods. In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5' end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G-U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion.

Aniline Compounds↗
PubMed1991

Interaction of RNA polymerase II with acetylated nucleosomal core particles.

Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery.

Acetic Anhydrides↗
PubMedDate not supplied

Kinetics of coupling reactions that generate monothiophosphate disulfides: implications for modification of RNAs.

The inclusion of guanosine-5'-monothiophosphate (GMPS) in an in vitro transcription reaction facilitates enzymatic synthesis of an RNA transcript with a monothiophosphate group at the 5' end. A kinetic study of the modification reactions that generate monothiophosphate disulfide linkages with either 5'-GMPS alone or 5'-GMPS-primed RNA as the substrate revealed that the second-order rate constants increased as the pH was decreased. For example, when the reaction pH was lowered from 8 to 4, the k2 value for the coupling reaction between N-(6-[biotinamido]hexyl)-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) and GMPS increased 67-fold from 1.84 to 123 M(-1) x s(-1). In addition to discussing a possible mechanism for coupling reactions that involve GMPS and disulfides, we also indicate conditions that are likely to be optimal for modification of the nucleophilic sulfur in 5'-GMPS-primed RNAs.

Cell-Free System↗
PubMed1991

Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase.

A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.

Base Sequence↗
PubMed2000

AVANTI 2. Randomized, double-blind trial to evaluate the efficacy and safety of zidovudine plus lamivudine versus zidovudine plus lamivudine plus indinavir in HIV-infected antiretroviral-naive patients.

OBJECTIVES: To investigate the effect of combination antiretroviral therapy on plasma HIV-1 RNA as measured by HIV RNA PCR and to assess their safety and tolerability. DESIGN: A randomized, multicentre, double-blind, placebo-controlled trial. SETTING: Multicentre study in eight European countries, Australia and Canada. PATIENTS: Antiretroviral naive patients (n = 103) with CD4 cell counts between 150 and 500 x 10(6)/l. INTERVENTION: Patients were randomly assigned to zidovudine (ZDV; 200 mg three times per day) plus lamivudine (3TC; 150 mg twice per day) or to ZDV + 3TC + indinavir (IND; 800 mg q8h) for 52 weeks. MAIN OUTCOME MEASURES: Degree and duration of reduction of plasma HIV-1 RNA as measured by RNA PCR; Development of drug-related toxicities sufficiently severe to warrant dose modification, interruption or permanent discontinuation. RESULTS: ZDV + 3TC + IND reduced plasma HIV-1 RNA (P < 0.001) and increased CD4 cell count significantly (P = 0.01) more than ZDV + 3TC. The addition of IND to ZDV + 3TC as initial therapy markedly increased the proportion of patients with plasma HIV-1 RNA values < 500 copies/ml (31/52, 60%) or 20 copies/ml (24/52, 46%) as compared with ZDV + 3TC (9/50, 18% or 2/50, 4% respectively) at week 52 in an intention-to-treat, missing = failure analysis. Assessment of time to virological rebound (> 0.5 log10 copies/ml above nadir) showed that patients who attained a minimum plasma HIV-1 RNA of < or = 20 copies/ml were less likely to rebound than those who did not reach this threshold. The addition of IND to ZDV + 3TC did not result in any significant increase in adverse experiences. CONCLUSION: ZDV + 3TC + IND resulted in a considerable improvement compared with the double combination, in reduction in plasma HIV-1 RNA, increase in CD4 cell count and proportion of patients with HIV RNA below the limit of detection. Despite an average 3 log10 decrease in plasma HIV-1 RNA on triple therapy, however, maximal suppression (< or = 20 copies/ml) was only attained in about one-half of the patients in an intent-to-treat analysis.

Adult↗
PubMed1982

Transcription initiation by RNA polymerase II is inhibited by S-adenosylhomocysteine.

Most eukaryotic mRNAs are blocked at their 5' termini by guanylylation and methylation. These "cap structures" have been shown to play important roles in increasing the stability and translatability of mRNAs. Previous in vitro and in vivo data suggest that these modifications occur extremely early in the synthesis of RNA transcripts by RNA polymerase II. Here we show that S-adenosylhomocysteine (AdoHcy), both a product and an inhibitor of transmethylation reactions, inhibits transcription initiation by RNA polymerase II, but not by RNA polymerase III, in a HeLa whole-cell lysate. AdoHcy must be present during initiation to inhibit transcription and does not affect elongation by RNA polymerase II or the stability of the resultant transcript. Furthermore, AdoHcy does not inhibit transcription by purified HeLa RNA polymerase II. These results suggest that formation of the 5'-cap structure is coupled to initiation of transcription and is consistent with a close association between the capping enzymes and RNA polymerase II at the time of initiation.

Adenoviruses, Human↗
PubMedDate not supplied

Eosinophilic lung inflammation in particulate-induced lung injury: technical consideration in isolating RNA for gene expression studies.

Particulate and other pollutant exposures are associated with lung injury and inflammation. The purpose of this study was to develop an approach by which intact RNA could be obtained from inflamed lung tissue from particulate-exposed animals in order to correlate injury with specific gene expression. Male Sprague Dawley (SD) and Fischer-344 (F-344) rats were intratracheally instilled with saline or residual oil fly ash (ROFA) particles, 8.3 mg/kg body weight in saline. At various time points following ROFA instillation, lungs were either lavaged or used for RNA isolation. ROFA exposure produced an increase in bronchoalveolar lavage fluid (BALF) neutrophils in both SD and F-344 rats. A time-dependent increase in eosinophils occurred only in SD rats but not in F-344 rats. Extraction of inflamed pulmonary tissue having a high influx of eosinophils for RNA using the conventional acid guanidinium thiocyanate phenol-chloroform (AGPC) procedure failed to provide undegraded RNA suitable for RT-PCR and Northern blot analysis of beta-actin mRNA expression. Mixing intact total RNA from saline control rat lungs with degraded RNA samples from inflamed lung yielded a gel profile of degraded RNA, indicating the presence of ribonuclease-like activity in the RNA extracted from lung tissues having eosinophil influx. Evidently, the conventional AGPC procedure failed to completely remove ribonuclease activity associated with ROFA-induced pulmonary eosinophil influx. This study reports a single-step modification to the AGPC extraction method that does not require additional reagents or additional precipitation steps for extracting undegraded RNA from nuclease-rich inflamed lung tissue. The aqueous layer resulting from mixing homogenate and chloroform is extracted a second time using an equal volume of AGPC buffer followed by addition of chloroform and centrifugation. The second aqueous phase is then treated as described in the conventional RNA extraction protocol. This simple and convenient modification does not require multiple precipitations of RNA and yields undegraded RNA from inflamed lung tissue with a slightly higher A260/A280 ratio without affecting overall RNA recovery. The results indicate that undegraded RNA could not be isolated using the routine AGPC-based isolation technique from lung tissue containing eosinophils following ROFA exposure. The degraded RNA preparations were unsuitable for gene expression studies. However, undegraded RNA can be isolated from these tissues by modifying the original AGPC RNA extraction procedure, which is suitable for gene expression analysis using northern blot and RT-PCR techniques.

Animals↗
PubMed1982

Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis.

RNA polymerase II was purified from Morris hepatoma 3924A by a series of ion-exchange and affinity column chromatographic fractionations, followed by sucrose gradient centrifugation in the presence of 0.3 M KC1. Purified RNA polymerase II had a specific activity of greater than 400 nmol of UMP incorporated (30 min)-1 (mg of protein)-1 by using double-stranded DNA as template. The purified enzyme contained five polypeptides (Mr 214 000, 140 000, 33 000, 25 000, and 21 000) that were present in molar quantities and two additional polypeptides (Mr 19 000 and 18 000) that had a combined molar ratio of 1.0. The cyclic AMP independent nuclear protein kinase NII, also purified from hepatoma 3924A, was able to phosphorylate RNA polymerase II polypeptides of Mr 214 000, 140 000, and 21 000. Phosphorylation of the polymerase was accompanied by enhanced transcription of double-stranded DNA, heat-denatured DNA, and poly[d-(A-T)]. The elevation in RNA polymerase activity was dependent upon the presence of hydrolyzable ATP and resulted from an increased number of RNA molecules synthesized in vitro. The average length of RNA chains was not affected by the kinase. Under similar conditions, protein kinase NII also stimulated homologous RNA polymerase I. In contrast to the phosphorylation of polymerase II, modification of polymerase I resulted in an increase in the average size, but not number, of RNA chains synthesized. The specificity of the NII kinase-catalyzed reaction was demonstrated by the inability of another homologous protein kinase, NI, to phosphorylate or activate RNA polymerase II.

Animals↗
PubMed1982

Q beta replicase containing altered forms of ribosomal protein S1.

Transcription of Q beta RNA replicase requires that the host-encoded Escherichia coli ribosomal protein S1 be present as a subunit of the replicase. To determine whether the activities of S1 in protein synthesis are operational in Q beta RNA transcription, we formed altered replicase enzymes by reconstituting replicase lacking S1 (R(-S1)) with modified S1 species whose properties in nucleic acid binding and protein synthesis had been previously established. S1 derivatized with N-ethylmaleimide reconstitutes a modified replicase that is 81% as active as replicase reconstituted with unmodified S1. S1 derivatized with N-ethylmaleimide and unmodified S1 bind with comparable affinity to R(-S1) (1 X 10(8) M-1). These results indicate that the helix-unwinding properties of S1, which are known to be inactivated by N-ethylmaleimide modification, are not required for Q beta RNA transcription and that the sulfhydryl-derivatized region of S1 is not utilized in replicase subunit contacts. In contrast with its established ability to replace E. coli S1 on the ribosome, Caulobacter crescentus S1 does not reconstitute Q beta RNA transcription activity when added to R(-S1). Our results suggest this inactivity may be due to poor binding to R(-S1).

Coliphages↗
PubMed1984

Isolation and partial characterization of messenger RNA, from murine T cell hybrids, coding for suppressive immunoglobulin G-binding factor.

Poly A RNA has been isolated from a murine T cell hybridoma ( T2D4 ) that spontaneously secretes suppressive immunoglobulin G-binding factor ( IgGBF ). Translation products, obtained from a rabbit reticulocyte lysate translation system and after injection into Xenopus laevis oocytes, contain material with the biologic activity, the affinity, and the m.w. of murine IgGBF ; it suppresses secondary in vitro IgG antibody production in a dose-dependent fashion. The suppressive factor binds to IgG but not to IgM immunoadsorbents and, after mild NaDodSO4 treatment, dissociates in NaDodSO4 polyacrylamide gels into two peaks at 78 and 40 kD. Translation products from two non- IgGBF -secreting cell lines (BW-5147, a T lymphoma line, and A9, a fibroblast cell line) fail to exert any suppressive activity. On sucrose gradients, the RNA responsible for the biologic activity was found in one major peak located at 11S. IgGBF synthesized in a cellfree translation system by using poly A RNA and sucrose gradient fractions was also characterized by immunoprecipitation with Fc fragments of [35S]methionine-labeled proteins. On NaDodSO4 polyacrylamide gels, it migrates in one peak located at 37 kD. We conclude that IgGBF is coded for by 11S poly A RNA and that no post-translational modifications (other than proteolytic cleavage) are necessary to obtain a biologically active factor with Ig-binding properties.

Animals↗
PubMed1978

Studies on the regulation of RNA synthesis in neuronal and glial nuclei isolated from rat brain.

In searching for regulatory mechanisms involved in the cell-specific neuronal and glial transcription a cell-free transcriptional system has been developed using neuronal and glial rat brain chromatin and partially purified neuronal and glial nuclear rat brain RNA polymerases. Both free and chromatin-bound (engaged) neuronal and glial RNA polymerase fractions were separated from isolated neuronal and glial rat brain nuclei to determine their transcriptive efficiency. A double number of RNA initiation sites was measured on the neuronal when compared to the glial chromatin, independently of whether the neuronal or the glial RNA polymerase preparation was used for the determination. Structural modification of the neuronal and glial chromatin template by acetylation with acetyl-coenzyme A leads to an increase of the total number of RNA initiation sites available for exogenously added rat brain RNA polymerase. This indicates that acetylation of chromatin-bound proteins is capable to render primarily restricted gene sequences transcriptable. A positive correlation exists between the extent of acetate uptake by neuronal and glial chromatin-bound histone fractions and the extent of the increase of the number of RNA initiation sites is specifically related to histone acetylation rather than to acetylation of any other chromatin protein. Significant information in this respect could be achieved by dissociation of chromatin into its principal components and selectively reconstituting DNA with specifically acetylated histone and non-histone proteins.

Animals↗
PubMed1997

Structural analysis of a necrogenic strain of cucumber mosaic cucumovirus satellite RNA in planta.

Structural studies of plant viral RNA molecules have been based on in vitro chemical and enzymatic modification. That approach, along with mutational analysis, has proven valuable in predicting structural models for some plant viruses such as tobacco mosaic tobamovirus and brome mosaic bromovirus. However, in planta conditions may be dramatically different from those found in vitro. In this study we analyzed the structure of cucumber mosaic cucumovirus satellite RNA (sat RNA) strain D4 in vivo and compared it to the structures found in vitro and in purified virions. Following a methodology developed to determine the structure of 18S rRNA within intact plant tissues, different patterns of adenosine and cytosine modification were found for D4-sat RNA molecules in vivo, in vitro, and in virions. This chemical probing procedure identifies adenosine and cytosine residues located in unpaired regions of the RNA molecules. Methylation data, a genetic algorithm in the STAR RNA folding program, and sequence alignment comparisons of 78 satellite CMV RNA sequences were used to identify several helical regions located at the 5' and 3' ends of the RNA molecule. Data from previous mutational and sequence comparison studies between satellite RNA strains inducing necrosis in tomato plants and those strains not inducing necrosis allowed us to identify one helix and two tetraloop regions correlating with the necrogenicity syndrome.

Base Sequence↗
PubMed1993

A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo.

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.

Animals↗
PubMed1979

Studies on the RNA and protein binding sites of the E. coli ribosomal protein L10.

We have used modification of specific amino acid residues in the E. coli ribosomal protein L10 as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of L10 was found to be inhibited by modification of one more of its arginine residues. This treatment does not affect the ability of L10 to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that L10's role in promoting the L7/L12-ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously.

Amino Acids↗
PubMed2002

The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2'-O-methyl modifications.

One of the problems that hamper the use of antisense DNAs as effective drugs is the non-specific binding of chemically-modified oligonucleotides to cellular proteins. We previously showed that the affinity of a model ssDNA-binding protein, the Ff gene 5 protein (g5p), was >300-fold higher for phosphorothioate-modified DNA (S-DNA) than for unmodified dA(36), consistent with the propensity of S-DNA to bind indiscriminately to proteins. The current work shows that g5p binding is also sensitive to sugar and pyrimidine modifications used in antisense oligomers. Binding affinities of g5p for 10 36mer oligomers were quantitated using solution circular dichroism measurements. The oligomers contained C-5-propyne (prC), 2'-O-methyl (2'-O-Me) or 2'-OH (RNA) groups, alone or combined with the phosphorothioate modification. In agreement with reported increases in antisense activity, the addition of prC or 2'-O-Me modifications substantially reduced the affinity of oligomers for g5p by approximately 2-fold compared with the same DNA oligomer sequences containing only phosphorothioate linkages. That is, such modifications moderated the propensity of the phosphorothioate group to bind tightly to the g5p. The Ff g5p could be a useful model protein for assessing non-specific binding effects of antisense oligomer modifications.

Circular Dichroism↗
PubMed1991

The absence of modified nucleotides affects both in vitro assembly and in vitro function of the 30S ribosomal subunit of Escherichia coli.

16S RNA of Escherichia coli lacking all post-transcriptional modifications and with 5'-termini of pppGGGAGA-, pppGAA-, pppAAA-, and pAAA- were prepared by in vitro transcription of appropriately engineered plasmids with T7 or SP6 RNA polymerases. These synthetic versions of 16S RNA were compared with natural 16S RNA for their ability to reconstitute 30S ribosomal subunits in vitro using varied conditions for both the isolation of the RNA and for reconstitution. Under all conditions studied, natural 16S RNA assembled correctly, as judged by velocity centrifugation comparison with an internal standard of native 30S particles, and the recovered ribosomes were 80-100% as active as native 30S ribosomes in initiation complex formation, P site binding of AcVal-tRNA, A site binding of Phe-tRNA, and formation of the first peptide bond. In contrast, all of the synthetic constructs including pAAA-, which has the same sequence as native 16S RNA, were only partially active in reconstitution and in the functional assays. We conclude that the lack of the 10 methylated nucleotides and/or the 2 pseudouridylate residues present in natural 16S RNA must be responsible for the reduced activity of the synthetic RNAs in ribosome assembly and function.

Centrifugation, Density Gradient↗