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PubMed1996

Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease.

A new viroid associated with an apple fruit disorder similar to, but more severe than, the dapple apple disease induced in some varieties by apple scar skin viroid (ASSVd) has been found. The new viroid, tentatively termed apple dimple fruit viroid (ADFVd), is a circular RNA of 306 nucleotides which adopts a quasi-rod-like conformation of minimum free energy. It contains the core nucleotides of the central conserved region (CCR) of the ASSVd group as well as the terminal conserved region (TCR) present in all members of the ASSVd and potato spindle tuber viroid (PSTVd) monophyletic groups. ADFVd has the highest sequence similarity with ASSVd and the 294 nucleotide citrus viroid CVd-IIIb sharing with the latter an almost identical left terminal domain. The right- and left-hand termini of ADFVd are formed by almost perfect duplications of sequences found in the CCR upper and lower strands, respectively, of PSTVd and closely related viroids.

Base Sequence↗
PubMed2004

Low-energy circular dichroism of 2-aminopurine dinucleotide as a probe of local conformation of DNA and RNA.

Circular dichroism is commonly used to investigate the conformations of nucleic acids. However, many biochemical processes implicate conformational changes of particular nucleotide residues within DNA or RNA that cannot be studied by this method, because the CD of these residues is buried in the total signal of the polynucleotide. Here, we report a method to study local conformations of DNA or RNA that is based on the use of the CD of 2-aminopurine (AP) residues as a probe. AP is readily incorporated into DNA in place of adenine and does not significantly alter DNA structure. Unlike adenine, AP is fluorescent and this property has been used for many years to investigate local nucleic acid structure. We show here that the CD spectrum of AP dinucleotide, (AP)(2), exhibits a positive CD band at 326 nm, a spectral region in which nucleic acids (and proteins) do not absorb. Our results show that the bases of (AP)(2) are stacked in a right-handed helical conformation. A low-energy CD band is also observed when this nucleotide dimer is incorporated into double-stranded DNA. Control experiments show that this signal comes from the stacking of adjacent AP residues. We have used this CD signal to provide information about the conformation of the AP dinucleotide at a defined position within single- and double-stranded nucleic acids.

2-Aminopurine↗
PubMed2026

circASbase: A Comprehensive Database of Alternative Splicing Events in circRNAs.

Although extensive evidence has underscored the critical role of alternative splicing (AS) in generating mature circular RNA (circRNA) isoforms and augmenting their functional diversity, a significant gap remains in the availability of specialized databases housing circRNA AS events. To bridge this gap, we develop circASbase, a pioneering and comprehensive database that catalogs 452,129 AS events in 884,047 full-length circRNAs from 581 samples across 13 species, and provides rich annotations to facilitate understanding the splicing regulation of circRNA. Our findings reveal substantial differences between circRNAs and linear transcripts regarding the distribution and occurrence of AS events, highlighting the unique regulatory landscape of circRNAs. These special splicing events result in functional differences of circRNAs by affecting internal ribosome entry sites, N6-methyladenosine sites, open reading frames, protein features, microRNA targets, and more. In summary, circASbase not only meets the urgent need of the research community for data repositories, but also represents a significant advancement in our understanding of circRNA biology. With its user-friendly interfaces and web-based visualization tools, circASbase is poised to become an indispensable resource for researchers exploring the regulatory mechanisms and functional roles of AS events in circRNAs. This database will continuously drive new insights and discoveries in the field, setting the stage for further advancements in circRNA research. circASbase is freely available at http://reprod.njmu.edu.cn/cgi-bin/circASbase/.

Alternative Splicing↗
PubMed1989

Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA.

Circular and multimeric forms of the satellite RNA of tobacco ringspot virus and their autolytic processing reactions are well known. They suggest replication models in which key elements are rolling circle transcription and the processing of the resulting multimeric RNA to generate the unit, 'monomeric' satellite RNA sequence. We prepared plasmids bearing two distinct sequences of the satellite RNA. Each was arranged to allow transcription of an oligoribonucleotide (r-oligo) of the polarity that is complementary to encapsidated satellite RNA. One sequence has the autolytic processing phosphodiester bond, ApG, and the other is located at a distance of about 150 nucleotide (nt) residues. The second r-oligo accomplished cleavage of the first, in a catalytic fashion. Analysis of truncated forms showed that 10 nt of the ApG junction-containing r-oligo and 46 of the endoribonucleolytic r-oligo were sufficient for recognition in the cleavage reaction. These results map the sequences involved in autolytic processing of the complementary polarity satellite RNA to two regions.

Base Sequence↗
PubMed1985

2' phosphomonoester, 3'-5' phosphodiester bond at a unique site in a circular viral RNA.

Solanum nodiflorum mottle virus (SNMV) RNA2 is a single-stranded, covalently closed circular molecule. RNase T2 or nuclease P1 digests of this RNA contain a minor nucleotide of unusual chromatographic and electrophoretic mobility. This nucleotide is resistant to further digestion by T2 or P1 ribonucleases, or by alkali, but is sensitive to venom phosphodiesterase digestion. Alkaline phosphatase digestion yields a product which is RNase T2 and P1 sensitive. The products of these various digests show that the minor nucleotide is a ribonuclease-resistant dinucleotide carrying a 2' phosphomonoester group with the core structure C2'p3'p5'A. This dinucleotide is found in a unique RNase T1 product of SNMV RNA2, thus establishing a unique location in the sequence for the 2' phosphomonoester group at residue 49. Identical results have been obtained with a second related virus. The phosphomonoester group probably results from the RNA ligation event by which the molecules were circularised.

Plant Viruses↗
PubMed1980

Some yeast mitochondrial RNAs are circular.

11S and 18S fractions of yeast mitochondrial RNAs, isolated by electrophoresis through agarose gels, have been found by electron microscopy to contain approximately 50% circular molecules. Circles in the 11S fraction have a contour length of 0.36 +/- 0.02 micron, which is approximately equal to the length of the majority of linear molecules also present. Circles in the 18S fraction have an average length of 0.78 +/- 0.11 micron. The size distribution is broader than for the 11S fraction, and we cannot exclude the possibility that more than one size class may be present. The 11S circular RNA forms circular R loops and RNA-DNA hybrids with DNA fragments of the oxi 3 region of mtDNA, which contains the structural gene for subunit 1 of cytochrome oxidase. As judged from the electron micrographs, the complete RNA participates in hybrid formation and the sequences coding for it appear to be continuous. Both 11S and 18S circles withstand treatment with DNAase and pronase. They are not eliminated by treatment with 1 M glyoxal in 50% formamide for 1 hr at 50 degrees C. We conclude that they are covalently closed. The function of the circular RNAs is unknown. They may be active as mRNAs, storage forms, or arise in a cut-and-splice process which generates mRNAs from longer transcripts.

Mitochondria↗
PubMed1995

Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P identified by in vitro selection.

Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P (P RNA) have been obtained by in vitro selection. The selection method involves cleavage of a circular RNA library by the P RNA, isolation of the linear cleavage product, and regeneration of circular RNA to allow amplification and multiple cycles of selection. The use of circular RNA ensures that potential substrates can be selected without restricted location of the cleavage site. Such a selection method has been used previously to isolate RNA motifs that undergo autolytic cleavage with Pb2+ [Pan, T., & Uhlenbeck, O. (1992) Biochemistry 31, 3887-3895]. The circular RNA pool after eight cycles of selection was cleaved by the B. subtilis P RNA as efficiently as a pre-tRNA(Phe) substrate, estimated to be more than 10 orders of magnitude better than the unselected RNA library. Kinetic analysis of individual variants showed that the kcat/KM of the selected RNA was up to 4-fold higher than that of the pre-tRNA(Phe). When cleavage was carried out with Escherichia coli P RNA, the selected RNA was 10-60-fold less reactive than the reaction of the pre-tRNA(Phe). Two distinct classes of variants are selected, both of which appear to differ significantly from the known P RNA substrates. Terminal truncation experiments suggest that a large number of nucleotides in the class I variants can be deleted without affecting the cleavage activity. The resulting minimal class I substrates contain a short stem-loop with no other apparent helical structures. The class II substrates are cleaved within a putative helical stem that is formed entirely by the primer sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1985

Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity.

Full-length double-stranded cDNA was prepared from purified circular RNA of two new Australian field isolates of citrus exocortis viroid (CEV) using two synthetic oligodeoxynucleotide primers. The cDNA was then cloned into the phage vector M13mp9 for sequence analysis. Sequencing of nine cDNA clones of isolate CEV-DE30 and eleven cDNA clones of isolate CEV-J indicated that both isolates consisted of a mixture of viroid species and led to the discovery of eleven new sequence variants of CEV. These new variants, together with the six reported previously, form two classes of sequence which differ by a minimum of 26 nucleotides in a total of 370 to 375 residues. These two classes correlate with two biologically distinct groups when propagated on tomato plants where one produces severe symptoms and the other gives rise to mild symptoms. Two regions of the native structure of CEV, comprising 18% of the total residues, differ between the sequence variants of mild and severe isolates. Whether or not both of these regions are essential for the variation in pathogenicity has yet to be determined.

Base Sequence↗
PubMed1988

Stability of RNA in developing Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA.

Synthetic capped RNA transcripts injected into fertilized eggs of Xenopus laevis have a half-life of 3-4 h. Addition of a long (approximately 200 nucleotide) poly(A) tail increases the half-life to 6-8 h which approaches the half-life of natural polyadenylated globin RNA injected into embryos. Since exonucleolytic action alone could account for the degradation of RNA, we tested whether circular RNA is stable after injection and find that circles are exceptionally stable (half-life greater than 40 h). After the midblastula transition, polyadenylated chloramphenicol transferase (CAT) mRNAs transcribed from injected plasmids have a half-life of 2.5 h. Insertion of a 1000 nucleotide 3' untranslated region from the Xhox-36 gene into the transcripts does not affect the half-life. In contrast to the finding that internal sequences do not affect stability, we find that sequences from the TFIIIA message reduce the half-life of CAT mRNA from 2.5 h to less than 30 min. We conclude that most RNAs are degraded exonucleolytically from the 3' end, but specialized internal sequences can greatly destabilize the RNA, possibly by acting as a site for an endonuclease.

Animals↗
PubMed1999

Circular dystrophin RNAs consisting of exons that were skipped by alternative splicing.

Exon skipping by alternative splicing and circular RNA formation are proposed to be interrelated events. Since multiple patterns of alternative splicing have been demonstrated in both the 5' and 3' regions of the dystrophin gene, the dystrophin transcript in skeletal muscle cells provides a model system in which this idea is tested. Nine circular RNAs that were expected to result from known exon skipping patterns in the 5' region of this gene were in fact identified, but three other circular RNAs expected to result from other known exon skipping reactions in this region could not be detected. The identification of two unexpected circular RNAs led to the discovery of two novel alternative splicing reactions. One circular RNA originating in the 3' region of the gene was identified but it lacked one small sized exon compared with the expected exon structure. Circular RNAs from the 5' region of the dystrophin gene could not be detected in Duchenne muscular dystrophy patients who have deletions of one or more exons in this segment of the gene, even though alternative splicing products were identified. These results showed that circular RNA formation is not necessarily linked to exon skipping and suggest that an undetermined factor regulates circular RNA formation.

Alternative Splicing↗
PubMed1998

The dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious RNA.

Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.

Base Sequence↗
PubMed1982

Stiffness of viroids and viroid-like RNA in solution.

The sedimentation coefficients of the potato spindle tuber viroid, four viroid-like RNAs from cadang-cadang-disease, circular RNA from velvet tobacco mottle virus, circular RNA from Solanum nodiflorum mottle virus and double stranded RNA5 from cucumber mosaic virus were measured in the analytical ultracentrifuge. The numbers of nucleotides of the RNA species varied between 246 and 670. The hydrodynamic models of rigid rods and flexible cylinders were applied for the interpretation of the sedimentation coefficients. Double-stranded RNA5 from cucumber mosaic virus with 335 basepairs fits the model of a rigid rod with an hydrated diameter of 29 A. Potato spindle tuber viroid and the four viroid-like RNA species of cadang-cadang-disease form a homologous series of flexible cylinders with a Kuhn's statistical length lambda-1 of 600 A. The circular RNA from the two viruses mentioned above are more flexibel than the viroids and viroid-like RNAs. The hydrodynamic interpretation is in accordance with thermodynamic data and secondary structure models. In two of the RNAs from cadang-cadang, cruciform structures would also be possible on the basis of the nucleotide sequence. The hydrodynamic data, however, favour clearly the extended structure over the cruciform.

Genes, Viral↗
PubMed2005

Recombinant viral RdRps can initiate RNA synthesis from circular templates.

The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage phi6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the phi6 RdRp could only use circular RNAs made from the 3'-terminal sequence of the phi6 genome.

Catalytic Domain↗
PubMed2000

Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure.

Viroids replicate through a rolling-circle mechanism in which the infecting circular RNA and its complementary (-) strand are transcribed. The precise site at which transcription starts was investigated for the avocado sunblotch viroid (ASBVd), the type species of the family of viroids with hammerhead ribozymes. Linear ASBVd (+) and (-) RNAs begin with a UAAAA sequence that maps to similar A+U-rich terminal loops in their predicted quasi-rod-like secondary structures. The sequences around the initiation sites of ASBVd, which replicates and accumulates in the chloroplast, are similar to the promoters of a nuclear-encoded chloroplastic RNA polymerase (NEP), supporting the involvement of an NEP-like activity in ASBVd replication. Since RNA folding appears to be kinetically determined, the specific location of both ASBVd initiation sites provides a mechanistic insight into how the nascent ASBVd strands may fold in vivo. The approach used here, in vitro capping and RNase protection assays, may be useful for investigating the initiation sites of other small circular RNA replicons.

Base Sequence↗
PubMed1995

Inverted repeats are necessary for circularization of the mouse testis Sry transcript.

Circular non-polyadenylated RNA molecules have been identified as stable transcription products of the human ETS-1 and mouse Sry genes. RNA circularization has been proposed to require two steps. The first step utilizes intramolecular base pairing to produce a transient stem-loop structure. The second step involves splicing a downstream donor splice site (DSS) to a now closely appositioned upstream acceptor splice site (ASS) within the loop. We demonstrate that the presence of long inverted repeats (IR) flanking the mouse Sry gene leads to the formation of the Sry circular transcript in cultured cells. Circularization requires the presence of both IR. As few as 400 complementary nt are necessary for this process. The presence of the IR does not significantly stimulate intermolecular annealing and trans-splicing in vivo.

Animals↗
PubMed1984

Yeast mitochondria contain a linear RNA strand complementary to the circular intronic bI1 RNA of cytochrome b.

bI1 RNA (excised from the first intron of the long form of the cytochrome b gene of Saccharomyces cerevisiae mitochondria) hybridizes with the two strands of a Bg/II-MboI DNA segment from this region. This fraction is resistant to digestions by DNase I and RNase T1 and disappears completely upon alkali hydrolysis. Strand-specific labeling of an intronic DNA fragment, cloned in pBR322 plasmid, was accomplished through the use of a T4 DNA polymerase. The purity of the probes was demonstrated by cloning an exon-intron fragment and labeling it by the same procedure; mRNA and pre-mRNA bands hybridized only with the transcribed DNA strand whereas bI1 RNA hybridized with the two strands under the stringent washing conditions employed (tm + 20 degrees C). Several experimental results argue against the possibility that the observation of two complementary bI1 RNA strands results from a partial self-complementarity of the RNA. A pre-mRNA intermediate from a box8 (G5046) mutant, still containing this intron, hybridizes only with the transcribed DNA strand of the pure intronic probe. The amount of the non-sense bI1 RNA strand is very low, in cells from two wild-type strains, relative to the sense RNA strand during the early stages of growth on glucose. It increases as the cells are released from glucose repression. bI1 RNA is resistant to RNase. Very little self-complementarity is seen by computer analysis of the sequence. Purified bI1 RNA is seen by electron microscopy under non-denaturing conditions as a mixture of double-stranded circular and linear molecules thus confirming the existence of the two complementary strands. The disappearance of all material following alkali hydrolysis demonstrates that these are indeed two RNA strands. Under fully denaturing conditions a mixture of single-stranded circular and linear molecules is seen as reported previously (Cell, 19, 321-329, 1980). We conclude that yeast mitochondria contain the two complementary bI1 RNA strands, one circular and the other linear. Considering a largely asymmetrical transcription of the mitochondrial genome in yeast and assuming that circularization of some intronic RNAs is part of RNA processing, we do not believe that the two strands are each a mixture of linear and circular molecules. The ratio of non-sense to sense bI1 RNA in a cytoplasmic petite mutant, A1B1, also varies according to growth conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1984

Conformational transitions in viroids and virusoids: comparison of results from energy minimization algorithm and from experimental data.

Viroids are single-stranded circular RNA molecules of 240 to 400 nucleotides which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but replicate only as genomic part of a plant virus. Their structure and structural transitions have been investigated by thermo-dynamic, kinetic and hydrodynamic methods. The special features of the sequences of these RNAs, which are the basis for their secondary structures and structural flexibility, are investigated with theoretical methods. A set of thermodynamic parameters for helix growth and loop formation is selected from the literature to calculate secondary structures and structural transitions of single-stranded RNAs. Appropriate modifications of the chosen parameter set are discussed. For calculations we used either Tinoco-plots and the model of "cooperative helices" or the Zuker-program based on the exact algorithm of Nussinov et al, or both. Calculations were done for viroids and virusoids. As both are single-stranded, circular RNAs we had to modify the Zuker-program as described in the appendix. Calculations are done for different viroids, i.e. potato spindle tuber, citrus exocortis, chrysanthemum stunt, coconut cadang-cadang, and avocado sunblotch, and for two virusoids, i.e. the circular RNAs of Solanum nodiflorum mottle virus, and velvet tobacco mottle virus. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but statistical sequences. The comparison of viroids, virusoids, and circular RNA or random sequences confirms the uniqueness of viroid structure.

Algorithms↗
PubMed1987

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon.

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.

Animals↗