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At least 1,657 records · Page 92Linked to original sources
PubMed1995

Accurate and efficient N-6-adenosine methylation in spliceosomal U6 small nuclear RNA by HeLa cell extract in vitro.

Human U6 small nuclear RNA (U6 snRNA), an abundant snRNA required for splicing of pre-mRNAs, contains several post-transcriptional modifications including a single m6A (N-6-methyladenosine) at position 43. This A-43 residue is critical for the function of U6 snRNA in splicing of pre-mRNAs. Yeast and plant U6 snRNAs also contain m6A in the corresponding position showing that this modification is evolutionarily conserved. In this study, we show that upon incubation of an unmodified U6 RNA with HeLa cell extract, A-43 residue in human U6 snRNA was rapidly converted to m6A-43. This conversion was detectable as early as 3 min after incubation and was nearly complete in 60 min; no other A residue in U6 snRNA was converted to m6A. Deletion studies showed that the stem-loop structure near the 5' end of U6 snRNA is dispensable for m6A formation; however, the integrity of the 3' stem-loop was necessary for efficient m6A formation. These data show that a short stretch of primary sequence flanking the methylation site is not sufficient for U6 m6A methyltransferase recognition and the enzyme probably recognizes secondary and/or tertiary structural features in U6 snRNA. The enzyme that catalyzes m6A formation in U6 snRNA appears to be distinct from the prolactin mRNA methyltransferase which is also present in HeLa nuclear extracts.

Adenosine↗
PubMed2004

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs.

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.

5' Untranslated Regions↗
PubMed1999

Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit.

We have calculated at 5.0 A resolution an electron-density map of the large 50S ribosomal subunit from the bacterium Haloarcula marismortui by using phases derived from four heavy-atom derivatives, intercrystal density averaging and density-modification procedures. More than 300 base pairs of A-form RNA duplex have been fitted into this map, as have regions of non-A-form duplex, single-stranded segments and tetraloops. The long rods of RNA crisscrossing the subunit arise from the stacking of short, separate double helices, not all of which are A-form, and in many places proteins crosslink two or more of these rods. The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy-atom-derivatized crystal. We have determined the structure of the translation-factor-binding centre by fitting the crystal structures of the ribosomal proteins L6, L11 and L14, the sarcin-ricin loop RNA, and the RNA sequence that binds L11 into the electron density. We can position either elongation factor G or elongation factor Tu complexed with an aminoacylated transfer RNA and GTP onto the factor-binding centre in a manner that is consistent with results from biochemical and electron microscopy studies.

Archaeal Proteins↗
PubMed2006

Tsix defective in splicing is competent to establish Xist silencing.

Dosage differences of X-linked genes between male and female mammals are compensated for by a mechanism known as X-inactivation, and the noncoding Xist gene plays a crucial role in this process. The expression of Xist is regulated in cis by its noncoding antisense gene, Tsix, whose transcripts (though a fraction of them stay unspliced), are processed like common proteincoding RNAs. It has been suggested that certain classes of sense-antisense pairs of RNA are causally involved in not only gene regulation but also higher order chromatin structure in various organisms. In fact, recent studies demonstrated that Tsix modulates Xist expression through modification of the chromatin structure. It is still unknown, however, whether the RNA product is important for the function of Tsix or whether the antisense transcription is sufficient. To obtain insight into this issue, we eliminated the splicing products of Tsix in the mouse and explored the effects of this elimination on Tsix-mediated Xist silencing. To our surprise, the Xist locus was stably repressed on the X carrying the splicing-defective Tsix allele. Moreover, the repressive chromatin configuration was properly established at the Xist locus. These unexpected results indicate that the splicing products are dispensable for Tsix-mediated Xist silencing.

Animals↗
PubMed2005

The role of reverse genetics in the development of vaccines against respiratory viruses.

Despite their significance, the only available vaccines against respiratory viruses are those for the prevention of influenza. Attempts have been made to produce vaccines against other respiratory viruses using traditional techniques, but have met with little success. Reverse genetics, although still a relatively new tool for the manipulation of negative-strand RNA viruses, has great potential for the preparation of vaccines against many of the common respiratory viruses. In the preparation of live vaccines, reverse genetics systems allow the direct modification of the specific regions in the genomes of negative-stranded RNA viruses concerned with attenuation; the ultimate goal is the introduction of site-specific mutations through a cDNA intermediate in order to develop strains with the requisite attenuation, antigenic and growth properties needed in a vaccine. These techniques can also be used to disarm potentially highly pathogenic viruses, such as emerging H5N1 avian influenza viruses, in order to facilitate large-scale preparation of viruses for use in inactivated vaccines under conditions of manufacturing safety. Before these vaccines become available, residual issues concerned with intellectual property rights to the technology and its application will need to be resolved.

Animals↗
PubMed1994

Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis.

We have investigated in detail the higher order structure of the genomic hepatitis delta virus (HDV) ribozyme using various base-specific chemical probes under native, semi-denaturing, and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethyl sulfate [which reacts with A (at N1) and C (at N3)] and a carbodiimide [which reacts with U (at N3) and G (at N1)]. In addition, for probing G residues (at N7), RNA samples were treated with NaBH4 and aniline after modification by treatment with dimethyl sulfate. The sites of modified positions were identified by primer extension analysis with reverse transcriptase. In general, our results are consistent with the proposed pseudoknot model of secondary structure, a model that is based on data from ribonucleolytic cleavage experiments. Our results provide clues to the identification of interacting bases in the HDV ribozyme. Furthermore, using this method we identified local conformational changes in several stem variants.

Aniline Compounds↗
PubMedDate not supplied

Fidelity in transcription assessed by DNA-dependent RNA polymerases.

The use of in vitro transcription of polynucleotides by prokaryotic and eukaryotic DNA-dependent RNA polymerases has proven of great usefulness in detecting mutagenic base modifications. Quite similar results are obtained with ribo- and deoxyribopolymers as templates, and with Mg2+ and Mn2+ at optimal concentrations. Recent studies have centered on polymers containing etheno C, as well as its hydrated form. These modifications are of particular interest, since they are the consequence of metabolised vinyl chloride, a proven human carcinogen.

Animals↗
PubMed1999

A chemogenetic approach to RNA function/structure analysis.

Almost two dozen nucleotide analogs have been synthesized with alpha-phosphorothioate-tagged triphosphates and utilized in an interference modification approach termed Nucleotide Analog Interference Mapping. This method has made it possible to determine the chemical basis of RNA function and structure, including the identification of new rules for RNA helix packing, the functional analysis of a binding site for monovalent metal ions within RNA and the characterization of the catalytic mechanism of RNA enzymes.

Animals↗
PubMed1980

Characterization of the non-permissive infection of rabbit cornea cells by vesicular stomatitis virus.

Rabbit cornea cells (RC-60) restrict the reproduction of vesicular stomatitis virus (VSV) (Thacore & Youngner, 1975). In cells infected with VSV alone, an inhibition in the synthesis of VSV genome RNA is observed. A number of parameters which could affect virus RNA synthesis have been examined: virus transcription and post-transcriptional modification, translation, modification of proteins and migration of the G protein to the surface of the cell; they all appear to be normal, although somewhat diminished, in the restricted system. In these cells, therefore, it is the replication of VSV RNA that is directly inhibited, although limited synthesis of both (+) and (-) strand genome length RNA does occur. When the cells are co-infected with rabbit poxvirus (RPV) as a helper virus, however, VSV production is normal. Our studies suggest that RPV plays a role in the maturation as well as in the replication of VSV RNA in RC-60 cells. Certain mutants of RPV have been found to lack the helper function and are unable to convert the RC-60 cells into a permissive host for VSV. These mutants should facilitate the elucidation of the mechanism by which RPV is able to overcome the restriction in these cells.

Animals↗
PubMed1976

[Modification of Schmidt-Tannhauser's method of bacterial nucleic acid fractionation].

It was revealed that in fractionation of nucleic acids by Schmidt-Tannhauser's method up to 90% of the DNA of bacteria was destroyed in the course of alkaline hydrolysis changing into the RNA fraction (the alkali-labile fraction of the DNA). The authors suggest a modification of the method based on the precipitation of the alkali-labile DNA fraction from the RNA fraction with ethanol. This modified method permits to record over 96% of all the cell DNA of bacteria.

Bacteria↗
PubMed2004

The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha.

Addition of N-acetylglucosamine (GlcNAc) is a ubiquitous form of intracellular glycosylation catalyzed by the conserved O-linked GlcNAc transferase (OGT). OGT contains an N-terminal domain of tetratricopeptide (TPR) repeats that mediates the recognition of a broad range of target proteins. Components of the nuclear pore complex are major OGT targets, as OGT depletion by RNA interference (RNAi) results in the loss of GlcNAc modification at the nuclear envelope. To gain insight into the mechanism of target recognition, we solved the crystal structure of the homodimeric TPR domain of human OGT, which contains 11.5 TPR repeats. The repeats form an elongated superhelix. The concave surface of the superhelix is lined by absolutely conserved asparagines, in a manner reminiscent of the peptide-binding site of importin alpha. Based on this structural similarity, we propose that OGT uses an analogous molecular mechanism to recognize its targets.

Acetylglucosamine↗
PubMed2004

Trafficking and functional expression of cardiac Na+ channels.

Voltage-gated Na(+)channels are essential for the amplitude and upstroke velocity of the cardiac action potential, which are important determinants for impulse propagation and impulse conduction velocity of throughout the working myocardium. Mutations in the major cardiac Na(+)channel gene SCN5A have been implicated in rare, familial forms of cardiac arrhythmias, namely LQT3, Brugada syndrome, progressive cardiac conduction disorder and sudden infant death syndrome. It is increasingly recognized that such mutations--apart from changing channel gating characteristics--may also be related to changes in channel protein trafficking and expression. Regulation of ion channel protein expression depends on a fine-tuned balance among various processes, such as gene transcription, RNA processing, protein synthesis, assembly and post-translational modification, the transport to the cell surface, the anchoring to the cytoskeleton, and regulation of endocytosis and controlled degradation of the protein. During the last decade, interest in factors that control the expression level of ion channels and mechanisms that are involved in targeting of channel proteins to specific sub-cellular and membrane domains is increasing. This review focuses on the current knowledge of mechanisms of cardiac Na(+) channel protein trafficking and expression in cardiomyocytes and its relation to Na(+)-channelopathies.

Action Potentials↗
PubMed2005

Potential roles for short RNAs in lymphocytes.

RNA interference (RNAi) is an ancient and evolutionarily conserved process. In some eukaryotes, RNAi silences parasitic genetic elements. In plants, RNAi serves as an immune system against RNA viruses and transgenes and in worms, RNAi silences transposons. In mammals, RNAi has yet unknown functions. However, emerging roles for short RNAs and the factors that interact with them in other eukaryotes include chromatin modification, DNA deletion and DNA methylation, which may provide clues to the roles for short RNA function in mammals. For example, antigen receptor expression in lymphocytes is a highly regulated process and although much is known about chromatin modification and DNA deletion in the immune system, several molecular details of chromatin regulation remain elusive. This review compares emerging roles for short RNA function to processes required for antigen receptor expression in mammalian lymphocytes and predicts that short RNAs direct events required for successful lymphocyte-restricted gene expression.

Animals↗
PubMed1998

The annealing of tRNA3Lys to human immunodeficiency virus type 1 primer binding site is critically dependent on the NCp7 zinc fingers structure.

The nucleocapsid protein NCp7 of the human immunodeficiency virus type 1 contains two zinc fingers of the CX2CX4HX4C type, flanked by several basic residues, and plays a major role in viral infectivity. Thus, NCp7 was shown to promote annealing of the tRNA3Lys to the primer binding site, a key step in reverse transcription. However, previous in vitro experiments were unable to clarify the role of the zinc fingers in this process, due to nucleic acid aggregation induced by the basic N- and C-terminal domains of NCp7. We show here that deletion of these sequences in (12-53)NCp7 strongly reduces the formation of aggregates and allows a direct visualization of the binary or ternary complexes between NCp7 and nucleic acids by gel electrophoresis. (12-53)NCp7 is able to induce hybridization of the 33P tRNA3Lys and the human immunodeficiency virus type 1 viral RNA-(77-257), which contains the primer binding site. Modification of the proximal zinc finger conformation in Cys23(12-53)NCp7 led to a large reduction in this hybridization process, while replacement of Trp37 by Leu in the distal zinc fingers resulted in a complete absence of annealing activity. These data account for the in vivo loss of viral infectivity following these mutations and emphasize the critical role of the structure of the zinc finger domain of NCp7. This could facilitate a rational approach to new antiviral agents directed toward NCp7.

Amino Acid Sequence↗
PubMed1984

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.

Actins↗
PubMed2000

Effects of histone tail domains on the rate of transcriptional elongation through a nucleosome.

The N-terminal tail domains of the core histones play important roles in gene regulation, but the exact mechanisms through which they act are not known. Recent studies suggest that the tail domains may influence the ability of RNA polymerase to elongate through the nucleosomal DNA and, thus, that posttranslational modification of the tail domains may provide a control point for gene regulation through effects on the elongation rate. We take advantage of an experimental system that uses bacteriophage T7 RNA polymerase as a probe for aspects of nucleosome transcription that are dominated by the properties of nucleosomes themselves. With this system, experiments can analyze the synchronous, real-time, single-passage transcription on the nucleosomal template. Here, we use this system to directly test the hypothesis that the tail domains may influence the "elongatability" of nucleosomal DNA and to identify which of the tail domains may contribute to this. The results show that the tail domains strongly influence the rate of elongation and suggest that the effect is dominated by the N-terminal domains of the (H3-H4)(2) tetramer. They further imply that tail-mediated octamer transfer is not essential for elongation through the nucleosome. Acetylation of the tail domains leads to effects on elongation that are similar to those arising from complete removal of the tail domains.

Amino Acid Sequence↗
PubMed1998

The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains.

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

Binding Sites↗
PubMed2004

LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.

Base Sequence↗