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At least 865 records · Page 48Linked to original sources
PubMed1989

In vitro 3' end processing and poly(A) tailing of RNA in Trypanosoma cruzi.

Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs. The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved. Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease. Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated. This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required. As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system. Several other modification activities specific for RNA 3' termini, including terminal nucleotide transferases, a tRNA CCA maturation activity, and a 3' exonuclease were also identified in these T. cruzi nuclear extracts.

Animals↗
PubMed2006

[Advances in the RNA-directed DNA methylation in plants].

The RNA-directed DNA Methylation (RdDM) is one type of epigenetic modification which was firstly discovered in plant. RdDM can directly cause DNA modifications of the genome through RNA-DNA interactions. In plant, both of RdDM and mRNA degradation induced by siRNA can silence sequence specific genes through RNA. They play very significant roles in chromosome rearrangement, defence of virus invasion, regulation of gene expression and many processes of plant development. However, the mechanisms of RdDM are still unclear. In this paper the basic characteristics of RdDM were briefly summarized and advances in studies on mechanisms of RdDM were reviewed. These include the kinds of DNA methyltransferases and their functional mechanisms in RdDM, the relationships between DNA methylation and chromatin modification, and important proteins involved in the RdDM process. In plants, RdDM may occur at both the transcriptional and post-transcriptionnal levels, both of which induce gene silencing. Methylation of the target gene promoter correlates with transcriptional gene silencing (TGS) whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS). RdDM and RNAi all depend on the similar siRNA and enzymes, such as DCL3, RdR2, SDE4 and AGO4. There are at least three kinds of DNA methyltransferases, DRM1/2, MET1 and CMT3, in pants. They can interact with and modifies all cytidines within the DNA regions homologous to RNA sequence. Furthermore, methylation of lysine 9 in Histone H3 can affect the methylation of cytidines.

DNA Methylation↗
PubMed1996

The use of chemical modification interference and inverse PCR mutagenesis to identify the dimerization initiation site of HIV-1 genomic RNA.

The retroviral genome consists of two identical RNA molecules physically linked together close to their 5' end, in a region called the Dimer Linkage Structure (DLS). Recent findings suggest that dimerization is involved in encapsidation, regulation of translation and reverse transcription. Previous in vitro studies localized the DLS of HIV-1 in a region downstream of the splice donor (SD) site. More recently, we showed that dimerization of HIV-1 RNA also involves sequences upstream of the SD site. Modification interference experiments and site-directed mutagenesis were used to identify the nucleotides required in the dimerization process of HIV-1 RNA. Our results point out a self-complementary sequence located in a hairpin loop, between the Primer Binding Site (PBS) and the SD site, as the Dimerization Initiation Site.

Base Sequence↗
PubMed2005

Protecting RNA in fixed tissue: an alternative method for LCM users.

RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.

Animals↗
PubMed2000

A post-translational modification of nuclear proteins, N(G),N(G)-dimethyl-Arg, found in a natural HLA class I peptide ligand.

Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self-tolerance and induction of cytotoxic T-cell responses against intracellular parasites. Despite frequent and diverse post-translational modification of eukaryotic cell proteins, very few class I-bound peptides with post-translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA-B39 (B*3910) derived from an RNA-binding nucleoprotein that carried N(G),N(G)-dimethyl-Arg. Although common among RNA-binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl-Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post-translationally modified ligand was a prominent component (1-2%) of the B*3910-bound peptide repertoire. The dimethyl-Arg residue was located in a central position of the peptide, amenable to interacting with T-cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post-translationally modified residue may have a major influence on the antigenic properties of this natural ligand.

Arginine↗
PubMed1983

Chemical modification of cytosine residues of mouse 5 S ribosomal RNA with hydrogen sulfide. (Nucleosides and nucleotides 43).

Cytosine residues of nucleic acids were converted to 4-thiouracil residues with hydrogen sulfide in pyridine and water to examine the secondary and tertiary structures of mouse 5 S rRNA. The cytosine residues at positions 10, 24, 34 (or 36), 39, 44 (or 46) and 63 were converted preferentially when the treatment was carried out at 28 degrees C. This result supports the model of the secondary structure of 5 S rRNA of Nishikawa, K. and Takemura, S. ((1974) FEBS Lett. 40, 106-109) consisting of five helices and five loops. As the temperature was increased to 35 degrees C, additional cytosine residues in positions 26, 52 and 78 were modified to moderate extents.

Animals↗
PubMed1987

Organothallium(III) reagents for modification of biomacromolecules: interaction of thallium derivatives with transfer RNA.

As an extension of work on the inhibition of enzymes by arylthallium(III) reagents, the thallium analogues of the organomercurials, we have studied the interactions of these molecules with transfer RNA. In contrast to thallous acetate, thallium(III) derivatives (thallic trifluoroacetate, p-methylphenylthallium(III) bis-trifluoroacetate (MPT) and o-carboxyphenylthallium(III) bis-trifluoroacetate) bound to Escherichia coli tRNA. The interaction was fully reversible upon Sephadex G-25 gel filtration, and binding constants and stoichiometries were evaluated by a number of procedures. The likely site of interaction was shown to be the thiouridine residue (s4U8) based on changes induced by MPT on the absorbance around 330 nm. No changes in stacking interactions could be detected from the absorption or circular dichroic spectra. The detailed structure of the groups on thallium(III) affected the interaction with tRNA. Thalliation at s4U8 affects the absorbance at 335 nm and the amino-acid uptake capacity of E. coli tRNAPhe in parallel, the latter being progressively inhibited by increasing amounts of MPT. In a model nucleoside system, uridine disulphide is probably formed from reduced thiouridine by the oxidative action of the Tl(III) reagents. No evidence of cross-linking of E. coli tRNA molecules under gel electrophoretic conditions was obtained in contrast to the model nucleoside. The easily reversible interaction of MPT with sulphur sites in E. coli tRNA contrasts with the stable (to gel filtration) bonds formed between MPT and (thiol) sites in enzymes.

Chromatography, Gel↗
PubMed2003

A novel, modification-dependent ATP-binding aptamer selected from an RNA library incorporating a cationic functionality.

An analogue of uridine triphosphate containing a cationic functional group was incorporated into a degenerate RNA library by enzymatic polymerization. In vitro selection experiments using this library yielded a novel receptor that binds ATP under physiological pH and salt conditions in a manner completely dependent on the presence of the cationic functionality. The consensus sequence and a secondary structure model for the ATP binding site were obtained by the analysis of functional sequences selected from a partially randomized pool based on the minimal parental sequence. Mutational studies of this receptor indicated that several of the modified uridines are critical for ATP binding. Analysis of the binding of ATP analogues revealed that the modified RNA receptor makes numerous contacts with ATP, including interactions with the triphosphate group. In contrast, the aptamer repeatedly isolated from natural RNA libraries does not interact with the triphosphate group of ATP. The incorporation of a cationic amine into nucleic acids clearly allows novel interactions to occur during the molecular recognition of ligands, which carries interesting implications for the RNA world hypothesis. In addition, new materials generated from such functionalized nucleic acids could be useful tools in research and diagnostics.

Adenosine Triphosphate↗
PubMed2003

Modification of the 5' terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication.

We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40 degrees C upon addition of AU, AUA, or AUU to the 5' terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3'-UA-5' is required at the 3' terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5' end of the genomic RNA provides unpaired 3'-UA-5' at the 3' end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3'-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3' terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

5' Untranslated Regions↗
PubMed1980

Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus.

Purified RNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex from vaccinia virus contains RNA triphosphatase activity. The latter activity, which removes the gamma-phosphate from triphosphate-ended polyribonucleotides, co-chromatographed with the capping and methylating enzyme complex on seven different ion exchange or affinity columns and co-sedimented with the complex on a glycerol gradient. On a molar basis, the RNA triphosphatase was about 100 times more active than the associated RNA guanylyl-transferase. When the purified enzyme complex was incubated with poly(A) containing a 5'-triphosphate, removal of the gamma-phosphate preceded capping. Furthermore, there was no significant difference in the rate or extent of capping 5'-diphosphate- or 5'-triphosphate-ended poly(A). Physical association of the three enzymatic activities appears to be an efficient mechanism for carrying out the following successive steps in cap formation: (formula: see text).

Acid Anhydride Hydrolases↗
PubMed1976

Modification of the 5' terminus of mRNA by an RNA (guanine-7-)-methyltransferase from HeLa cells.

The 5' termini of many viral and cellular mRNAs contain sequences of the type m7G(5")pppNm. An RNA (guanine-7-)-methyltransferase that specifically methylates the 5'-terminal guanosine residue of RNAs ending in the dinucleoside triphosphate G(5')pppN- has been purified from the cytoplasm of HeLa cells. Approximately two-thirds of the methyltransferase activity detected in an assay employing umnethylated vaccinia virus mRNA as acceptor was located in the cytoplasm when cells were disrupted by Dounce homogenization; 30% of the cytoplasmic activity was associated with ribosomes but was removed by washing with 0.5 M KCl. The enzyme was purified 165-fold from the cytoplasm by removing nucleic acid by phase partition followed by ammonium sulfate precipitation and column chromatography on DEAE-cellulose, denatured DNA-agarose, and CM-Sephadex. The partially purified enzyme preparation methylated heterologous tRNAs as well as vaccinia mRNA, but the tRNA methyltransferases could be separated from the mRNA activity by sucrose gradient sedimentation and gel filtration on Sephadex G-200. The product of the partially purified enzyme using vaccinia mRNA as substrate was exclusively 7-methylguanosine located in the terminal dinucleoside triphosphate. In addition to RNAs and synthetic polyribonucleotides terminating in a dinucleoside triphosphate, free G(5')pppG could be methylated but GTP, GDP, and G(5')pppG could not. The enzyme also methylated the dinucleoside diphosphate G(5')pppG but much less efficiently than G(5')pppG. An S20, W of 3.8, a Stokes radius of 3.6 nm, and a molecular weight of 56,000 were obtained from sucrose gradient sedimentation and Sephadex G-200 column chromatography.

Base Sequence↗