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At least 109 records · Page 6Linked to original sources
PubMed2002

Chimeric RNA transposition intermediates of the I factor produce precise retrotransposed copies.

I elements in Drosophila melanogaster are non-long terminal repeat (LTR) retrotransposons of particular interest because high levels of transposition can be induced by appropriate crosses. They use a full-length RNA transposition intermediate as a template for reverse transcription. Detailed molecular characterization of this intermediate is rendered difficult because of the many transcripts produced by defective elements. The use of an active I element marked with a sequence encoding the HA epitope solves this problem. We used an RNA circularization procedure followed by RT-PCR to analyze the transcripts produced by actively transposing tagged I elements. Most start at the 5' end at the second nucleotide of the I element and all are polyadenylated at a site located in genomic sequences downstream of the 3' end. One of the tagged I elements, inserted in locus 88A, produces chimeric transcripts that carry sequences from both 5'- and 3'-flanking genomic DNA. We show that synthesis of these chimeric transcripts is controlled by the I element itself. Analysis of full-length transposed copies of this element shows that the extra sequences at the 5' and 3' ends are not integrated during retrotransposition. This suggests that initiation and arrest of reverse transcription during retrotransposition are precise processes.

3' Flanking Region↗
PubMed1993

The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates.

Human hepatitis delta virus has a single-stranded circular RNA genome that replicates by RNA-directed RNA synthesis. The virus encodes only a single protein, the delta antigen, which both is small (22 kDa) and lacks sequence homology to known RNA polymerases, suggesting that the virus employs a cellular polymerase for replication. Consistent with this suggestion, we have used homogenized nuclei from a human hepatoma cell line, HepG2, to demonstrate RNA-directed RNA synthesis from both genomic hepatitis delta virus RNA and its complement, the antigenomic RNA. RNA polymerase II was responsible for this transcription because the reaction was inhibited both by low doses of alpha-amanitin and by a monoclonal antibody specific for polymerase II. In addition, it was found that the majority of the RNA products were processed, presumably by self-cleavage and self-ligation, to produce covalently closed circular molecules.

Amanitins↗
PubMed1993

Endogenous promoters can direct the transcription of hepatitis delta virus RNA from a recircularized cDNA template.

Transcription and replication of hepatitis delta virus (HDV) RNA is thought to be performed by host RNA polymerase II. The mechanism which enables polymerase II to use RNA as a template is unclear. However, since extensive intramolecular complementarity allows HDV RNA to form a rod-shaped structure, it is possible that the mostly double-stranded HDV RNA may resemble double-stranded DNA in structure, and can thus be used by RNA polymerase II as a template. To investigate this possibility, we examined whether the cDNA counterpart of HDV RNA contains a promoter and thus can drive the transcription and replication of HDV RNA. Circularized monomers of HDV cDNA, when transfected into various cell lines, were found to generate both monomeric and dimeric forms of HDV RNA and hepatitis delta antigen at levels comparable to those generated with HDV cDNA multimers under the control of a SV40 late promoter, suggesting that HDV cDNA contains endogenous promoters. Using chloramphenicol acetyltransferase and human growth hormone as reporter genes, the specific promoter activity for the synthesis of antigenomic HDV RNA was localized to a 29-nucleotide region (nucleotides 1650-1679), although an additional 224-nucleotide upstream region was also necessary for maximum activity. Similarly, promoter activity for the synthesis of genomic RNA was localized to a 160-nucleotide region around position 1679 that overlapped with the antigenomic promoter region. Since these regions are in a highly conserved double-stranded region of HDV RNA, they may represent RNA promoters recognized by RNA polymerase II. This result also suggests a convenient method, using circularized monomer HDV cDNA, to study HDV RNA replication.

Base Sequence↗
PubMed1991

Is 20S RNA naked?

The 20S RNA of Saccharomyces cerevisiae is a single-stranded, circular RNA virus. A previous study suggested that this RNA is part of a 32S ribonucleoprotein particle, being associated with multiple copies of a 23-kilodalton protein. We show here that this protein is, in fact, the chromosome-encoded heat shock protein Hsp26. Furthermore, it is apparently not associated with 20S RNA and plays no obvious role in the life cycle of the virus.

Centrifugation, Density Gradient↗
PubMed1995

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.

Base Sequence↗
PubMed1977

The genome of Uukuniemi virus consists of three unique RNA segments.

The three RNA species isolated from virions of Uukuniemi virus, a proposed member of the newly defined Bunyaviridae family, have been characterized by analysis of 32P-labeled ribonuclease T1 oligonucleotides separated on two-dimensional polyacrylamide gels. Each species contains unique oligonucleotides not present in the two others, indicating that the genome of this virus is segmented. Each segment appears to contain a unique primary sequence with little or no overlapping among the segments. The complexities of the RNA segments as calculated from the radioactivity in unique oligonucleotides of defined lengths are about 8000 (L RNA), 3500 (M) and 1900 (S) nucleotides. Since these values are similar to the molecular weights determined by other methods, each size class of RNA corresponds to a single molecular species. The presence of a 5' terminal pppAp ... structure in each RNA segment confirms indications from electron microscopy that the apparently circular RNA segments are not covalently closed. The absence of either a 5' terminal "cap" or 3' terminal poly(A) supports the concept that Uukuniemi virus is a negative strand virus.

Arboviruses↗
PubMed2000

Origin of hepatitis delta virus mRNA.

Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5' end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5'-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5' ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5' end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3'-end addition to the template. These new findings support the interpretation that the 5' end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.

5' Untranslated Regions↗
PubMed1989

Human hepatitis delta virus RNA subfragments contain an autocleavage activity.

Hepatitis delta virus (HDV) contains a single-stranded circular RNA genome of 1.7 kilobases. In this report we demonstrate that subfragments of HDV RNA can undergo autocatalytic cleavage. This cleavage requires at least 500 microM of Mg2+ or Ca2+, is not affected by varying the pH from 5.0 to 9.1, and occurs with RNA fragments as small as 133 nucleotides. The larger RNA fragments containing additional HDV sequences have a lower efficiency of cleavage. Deletion analysis at both ends of RNA subfragments suggested that the catalytic ability of HDV RNA resides in a stretch of no more than 117 nucleotides around the cleavage site. The cleavage occurs at the phosphodiester bond between nucleotides 688 and 689 on the HDV genomic map, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3' fragment with a guanosyl residue with a 5'-hydroxyl group. The smallest autocleaving RNA does not contain the "hammerhead" sequence required for the autocleavage of other known self-cleaving RNA. The cleavage of HDV RNA occurs at a much faster rate, even at a very low Mg2+ concentration, than that of other "ribozymes." Thus, HDV RNA represents a distinct class of ribozyme.

Base Sequence↗
PubMed1993

Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites.

Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.

Animals↗
PubMed1984

A labile phosphodiester bond at the ligation junction in a circular intervening sequence RNA.

The excised intervening sequence of the Tetrahymena ribosomal RNA precursor mediates its own covalent cyclization in the absence of any protein. The circular molecule undergoes slow reopening at a single phosphodiester bond, the one that was formed during cyclization. The resulting linear molecule has 5'-phosphate and 3'-hydroxyl termini; these are unusual products for RNA hydrolysis but are typical of the other reactions mediated by this molecule. The reopened circle retains cleavage-ligation activity, as evidenced by its ability to undergo another round of cyclization and reopening. The finding that an RNA molecule can be folded so that a specific phosphate can be strained or activated helps to explain how the activation energy is lowered for RNA self-splicing. The proposed mechanisms may be relevant to several other RNA cleavage reactions that are RNA-mediated.

Animals↗
PubMed2005

Viroids and viroid-host interactions.

Although they induce symptoms in plants similar to those accompanying virus infections, viroids have unique structural, functional, and evolutionary characteristics. They are composed of a small, nonprotein-coding, single-stranded, circular RNA, with autonomous replication. Viroid species are clustered into the families Pospiviroidae and Avsunviroidae, whose members replicate (and accumulate) in the nucleus and chloroplast, respectively. Viroids replicate in three steps through an RNA-based rolling-circle mechanism: synthesis of longer-than-unit strands catalyzed by host RNA polymerases; processing to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes; and circularization. Within the initially infected cells, viroid RNA must move to its replication organelle, with the resulting progeny then invading adjacent cells through plasmodesmata and reaching distal parts via the vasculature. To carry out these movements, viroids must interact with host factors. The mature viroid RNA could be the primary pathogenic effector or, alternatively, viroids could exert their pathogenic effects via RNA silencing.

Host-Parasite Interactions↗
PubMed1994

Specific RNA binding by amino-terminal peptides of alfalfa mosaic virus coat protein.

Specific RNA-protein interactions and ribonucleoprotein complexes are essential for many biological processes, but our understanding of how ribonucleoprotein particles form and accomplish their biological functions is rudimentary. This paper describes the interaction of alfalfa mosaic virus (A1MV) coat protein or peptides with viral RNA. A1MV coat protein is necessary both for virus particle formation and for the initiation of replication of the three genomic RNAs. We have examined protein determinants required for specific RNA binding and analyzed potential structural changes elicited by complex formation. The results indicate that the amino-terminus of the viral coat protein, which lacks primary sequence homology with recognized RNA binding motifs, is both necessary and sufficient for binding to RNA. Circular dichroism spectra and electrophoretic mobility shift experiments suggest that the RNA conformation is altered when amino-terminal coat protein peptides bind to the viral RNA. The peptide--RNA interaction is functionally significant because the peptides will substitute for A1MV coat protein in initiating RNA replication. The apparent conformational change that accompanies RNA--peptide complex formation may generate a structure which, unlike the viral RNA alone, can be recognized by the viral replicase.

Alfalfa mosaic virus↗
PubMed1988

Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications.

cDNA prepared from the single-stranded circular RNA genome of hepatitis delta virus was cloned in lambda gt11 by using RNA from the liver of an infected woodchuck. From the sequence of overlapping clones, we assembled the full sequence of 1,679 nucleotides. The sequence indicated an exceptional ability for intramolecular base pairing, yielding a rod structure with at least 70% of the bases paired and a predicted free energy of -805 kcal (-3,368 kJ)/mol. Three of the lambda clones contained sequences that were not only expressed as fusion proteins with beta-galactosidase but were recognized by human hepatitis delta virus-specific antibody. These clones were sequenced so as to establish the reading frame of the delta antigen on the antigenomic strand. The fusion protein produced by one clone was purified by immunoaffinity chromatography and then was used to raise rabbit antibodies specific for the delta antigen.

Animals↗
PubMed1983

A circular dichroism study of the structure of Penicillium chrysogenum mycovirus.

We have examined the absorption and circular dichroism spectra of intact Penicillium chrysogenum virus, empty capsid particles, and isolated double-stranded RNA. The absorbance at 260 nm of intact virus was less than 4% hypochromic relative to the absorbances of the free double-stranded RNA and free viral protein, indicating very little change in the base stacking interactions of the RNA. Circular dichroism studies of intact virus indicate that the capsid protein consists of 45% alpha-helix. Empty capsids, containing a protein of the same molecular weight as intact virus protein, were found to have 30% alpha-helix, suggesting a conformational change in the capsid upon assembly with RNA. The conformation of double-stranded RNA in the virus was slightly altered from the solution structure of the RNA in 0.01 M Na+ and resembled the conformation of double-stranded RNA partially bound with spermidine. However, the virus does not appear to contain polyamines. Electrophoretic experiments indicate a pH- and salt-titratable RNA binding site on the capsid protein in virus disrupted by urea or non-ionic detergents. The results are consistent with significant ionic interactions between the RNA and the capsid protein in the virus.

Capsid↗
PubMed1984

Splicing of large ribosomal precursor RNA and processing of intron RNA in yeast mitochondria.

We have studied splicing of precursors to the large ribosomal RNA and processing of the excised intron in yeast mitochondria using primer extension with reverse transcriptase and electron microscopy. Structural features of the following intermediates are described: first, a linear RNA carrying a 5'-terminal G that is not encoded in mitochondrial DNA; second, a circular RNA in which the 3' and 5' intron borders are covalently linked. Three nucleotides of the 5' intron border are absent from the site of circle closure. The properties of these intermediates fit remarkably well into the mechanism of self-splicing described for the ribosomal precursor RNA from Tetrahymena nuclei. A new feature of the yeast mitochondrial system is that the excised intron can have one of two destinies, circularization or cleavage at an internal position.

Base Sequence↗
PubMed2006

An efficient thermally induced RNA conformational switch as a framework for the functionalization of RNA nanostructures.

RNA offers a variety of interactions and dynamic conformational switches not available with DNA that may be exploited for the construction of nanomolecular structures. Here, we show how the RNA loop-loop, or "kissing", interaction can be used to construct specific circular RNA arrangements that are capable of thermal isomerization to alternative structures. We also show how this thermally induced structural rearrangement can be used to unmask a functional RNA structure, in this case, a peptide-binding RNA structure, the Rev-response element (RRE) of HIV, thereby acting as a functional peptide-binding switch. The relative ease with which the RRE could be engineered into the RNA substrates suggested that a variety of functional RNA structures may be introduced. In addition, the structural rearrangement was extremely efficient, showing that the "kissing" complexes described in this study may provide a useful framework for the construction of functional RNA-based nanostructures, as well as aid in our understanding of the way RNA functions in biological systems.

Base Sequence↗
PubMed1988

Self-cleaving viroid and newt RNAs may only be active as dimers.

Avocado sunblotch viroid (ASBV) is a 247-nucleotide, single-stranded, circular RNA. It is considered to replicate via a rolling-circle mechanism in which circular, monomeric plus and minus RNAs act as templates for the synthesis of longer-than-unit-length precursor RNAs. Processing of these RNAs in vivo may occur by a self-cleavage reaction, as indicated by ability of dimeric, linear plus and minus ASBV RNAs to specifically self-cleave in vitro with the excision of a monomeric RNA with 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. A similar self-cleavage reaction has also been reported to occur in an RNA transcript containing a dimeric copy of a tandemly repeated, 330-base-pair sequence of the newt genome. Based on comparisons with self-cleaving plant viral satellite RNAs, hammerhead-shaped active structures, each containing one self-cleavage site, were proposed for the plus and minus ASBV RNAs and the newt RNA, but the stability of these hammerheads has been questioned. Here, more stable active structures that contain two self-cleavage sites are proposed and data supporting these models are presented.

Animals↗