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[Kinetic method for the determination of sequences of purine and pyrimidine blocks for circular nucleic acids].

We prove that the sequence of purine and pyrimidine blocks can be restored for circular nucleic acids if molecular weight distributions of fragments of various stages of hydrolysis are known. Hydrolysis of nucleic acid is considered as a progressive fragmentation of a "ciphering", an arbitrary circular polymer, monomers of which have weights from 1 to 6 (corresponding to the real number of purines or pyrimidines in the block), while the site of fragmentation is selected at random. The hydrolysis of circular RNA under the action of pancreatic RNase proceeds in accordance with the model of ciphering. Heuristic method for restoration of the monomer sequence in the ciphering based upon some characteristics of molecular weight distributions (such as Sj(m)-number of the fragments with the weight m containing j unbroken bonds) is considered. Uniqueness of restoration of the monomers sequence of the ciphering based upon known set [Sj(m)] is proved.

Animals↗
PubMed1996

RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end.

Genome analogs ("minigenomes") of Sendai and measles viruses replicate efficiently only if their nucleotide length is an even multiple of six, a requirement called the rule of six (P. Calain and L. Roux, J. Virol. 67:4822-4830, 1993; M. S. Sidhu, J. Chan, K. Kaelin, P. Spielhofer, F. Radecke, H. Schneider, M. Masurekar, P. C. Dowling, M. A. Billeter, and S. A. Udem, Virology 208:800-807, 1995). The existence of a comparable requirement was tested for respiratory syncytial virus (RSV), which also is a member of family Paramyxoviridae and whose natural genome length also is a multiple of six. An internally truncated analog of RSV positive-sense replicative intermediate RNA (antigenome) bearing the chloramphenicol acetyltransferase gene as a reporter was synthesized from cDNA in vitro. This RNA was transfected into cells which were infected with RSV as a helper. Miniantigenomic RNA was indistinguishable from previously studied negative-sense minigenome RNA in its ability to participate in transcription, RNA replication, and incorporation into transmissible particles. Sixteen miniantigenomes which were of slightly different lengths and which in aggregate represented multiples of a wide range of integers including 1 to 15 were constructed. During transfection and two serial passages, the various miniantigenomes were essentially indistinguishable with regard to the efficiency of transcription, RNA replication, and packaging into transmissible particles. Progeny minigenomes of six different mutants were recovered postpassage, copied into cDNA, cloned, and sequenced completely. The length of each of these RNAs was found to have remained unchanged during replication and passage. Thus, RSV transcription and replication appear to lack the requirement that the template length be an even multiple of an integer such as six, which for Sendai and measles viruses is obligatory for nucleocapsid function. Each of the in vitro-synthesized miniantigenomes used in transfection contained a nonviral extension of three nucleotides, GGG, on the 5' (leader) end contributed by the T7 promoter. The termini of the recovered minigenomes were examined for five mutants by RNA circularization followed by cDNA synthesis, amplification, cloning, and sequencing. Unexpectedly, each recovered minigenome contained the complement of this nonviral extension on the 3' (leader) end, showing that it had been faithfully copied and maintained during RNA replication and passage. The nonviral trinucleotide did not appear to affect the activity of the template.

DNA, Complementary↗
PubMed1996

RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase.

Hepatitis delta virus (HDV) is a subviral human pathogen that requires hepatitis B virus (HBV) for packaging. Concurrent infection by HBV and HDV increases the risk of severe liver disease compared to infection with HBV alone. The HDV genome is a closed circular RNA of about 1,700 bases which is replicated through an RNA intermediate, the antigenome. Both RNAs can be folded into highly base-paired, rod-shaped structures, similar to the plant viroid RNAs. Two forms of the sole HDV protein, hepatitis delta antigen, are derived from a single open reading frame by RNA editing; the enzymes responsible for the editing have not been characterized. Here we report that the purified enzyme dsRAD (for double-stranded-RNA-adenosine deaminase) can edit HDV antigenomic RNA in vitro. Most important, we observe that mutations in critical sequences of the antigenome have identical effects on in vitro and in vivo editing, suggesting that dsRAD, or a closely related enzyme, is responsible for editing HDV RNA in vivo.

Adenosine↗
PubMed2004

Characterization of RNA-binding properties of three types of RNA-binding proteins in Anabaena sp. PPC 7120.

The Rbp proteins in cyanobacteria are RNA-binding proteins with a single RNA recognition motif or RRM. A comprehensive assembly of genomic data suggests that there are two major classes of Rbp proteins (classes I and II) that diverged before the diversification of cyanobacteria. Class I proteins are further classified into two types with or without a C-terminal glycine-rich domain. The results of selection from a random RNA pool suggest that RbpA1 (class I) has affinity to C-rich and G-rich sequences. In vitro RNA binding assay with homopolymers indicated that class II protein has low affinity to poly(G) in contrast with class I proteins. Site-specific mutagenesis analysis of the RRM in RbpA1 showed that the aromatic residues Tyr4 or Phe46 are important in RNA binding as well as maintenance of secondary structure. We also tested various truncated proteins lacking the C-terminal domain as well as point mutants. Most of these proteins exhibited decreased affinity to RNA. Circular dichroism analysis as well as chromatographic analysis showed that Tyr4 and Phe46 are also important in maintaining the structure of RbpA1 protein. The C-terminal glycine-rich domain itself does not contribute much to the RNA-binding, but Arg83 which is located close to the C-terminal end of RRM is important in the RNA-binding.

Amino Acid Sequence↗
PubMed1994

Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens.

Hepatitis delta virus (HDV) is composed of four specific components. The first component is envelope protein which contains hepatitis B surface antigens. The second and third components are nucleocapsid proteins, referred to as small and large hepatitis delta antigens (HDAgs). The final component is a single-stranded circular RNA molecule known as the viral genome. In order to study the mechanism of HDV RNA packaging, a four-plasmid cotransfection system in which each viral component was provided by a separate plasmid was employed. Virus-like particles released from Huh-7 cells receiving such a cotransfection were found to contain HDV RNA along with three proteins. Therefore, the four-plasmid cotransfection system could lead to successful HDV RNA packaging in vitro. The system was then used to show that the large HDAg alone was able to achieve a low level of HDV RNA packaging. Analysis of a variety of large HDAg mutants revealed that the RNA-binding domain was essential for viral RNA packaging. By increasing the incorporation of small HDAg into virus-like particles, we found a three- to fourfold enhancement of HDV RNA packaging. This effect was probably through a direct binding of HDV RNA, independent from that of large HDAg, with the small HDAg. The subsequent RNA-protein complex was packaged into particles. The results provided insight into the roles and functional domains of small and large HDAgs in HDV RNA packaging.

Antigens, Viral↗
PubMed2025

KDM3B Regulates Postradiation Fibrotic Responses in Prostate Stroma via N6-methyladenosine Modification of LOX.

PURPOSE: Genome-wide association studies have uncovered single-nucleotide polymorphisms (SNPs) linked to radiation therapy (RT)-induced toxicities in patients with prostate cancer. SNP rs17599026, located in intron 21 of the KDM3B gene, has been associated with late-onset urinary toxicity, with an increased frequency of urination observed 2 years post-RT compared with pretreatment conditions. This study aimed to explore the underlying mechanisms driving this association. METHODS AND MATERIALS: A clustered regularly interspaced short palindromic repeats-dead Cas9 prime editing system was used to mimic KDM3B genetic variants in prostate stromal cell lines. Murine models with wild-type and heterozygous Kdm3b genotypes were used to assess fibrosis following radiation. RNA immunoprecipitation, transcript stability assays, and protein analysis elucidated the role of N6-methyladenosine (m6A) modification in regulating lysyl oxidase (LOX) expression. α-ketoglutarate (α-KG) supplementation was tested for its effects on KDM3B protein stability, LOX expression, and fibrosis mitigation. RESULTS: The rs17599026 SNP reduced KDM3B protein expression via circular RNA and microRNA-mediated mechanisms, leading to decreased m6A modification and increased stability of LOX messenger RNA. Elevated LOX expression promoted collagen cross-linking and fibrosis in prostate stroma. α-KG supplementation restored KDM3B protein levels, reduced LOX expression, and mitigated fibrosis in vitro and in vivo. CONCLUSIONS: KDM3B genetic variations influence radiation-induced fibrosis through posttranscriptional regulation of LOX. Dietary α-KG supplementation may serve as a mechanism-based strategy to alleviate radiation toxicity in patients with prostate cancer, offering a potential therapeutic pathway to improve treatment outcomes.

Male↗
PubMed2001

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study.

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

Calorimetry↗
PubMed1985

Computer program for prediction of the optimal and suboptimal secondary structures of long RNA molecules.

We present an algorithm for prediction of RNA secondary structures. The program consists of three parts: the first computes location and free energy of every possible stem-loop structure, the second computes probability of its formation, and the third lists the positions and free energies of all the stem-loops in the order of their probability sizes. The circular RNA molecule of chrysanthemum stunt viroid was used as an input data for demonstrating the operation of the program.

Algorithms↗
PubMed2001

Natural 2',5'-phosphodiester bonds found at the ligation sites of peach latent mosaic viroid.

Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2',5'-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2', 5'-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2',5'-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2',5'-phosphodiester bonds are still present in nature and that they are of biological importance.

Mosaic Viruses↗
PubMed2003

Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action.

In animal cells, small interfering RNAs (siRNA), when exogenously provided, have been reported to be capable of inhibiting replication of several different viruses. In preliminary studies, siRNA species were designed and tested for their ability to act on the protein expressed in Huh7 cells transfected with DNA-directed mRNA constructs containing hepatitis delta virus (HDV) target sequences. The aim was to achieve siRNA specific for each of the three RNAs of HDV replication: (i) the 1,679-nucleotide circular RNA genome, (ii) its exact complement, the antigenome, and (iii) the less abundant polyadenylated mRNA for the small delta protein. Many of the 16 siRNA tested gave >80% inhibition in this assay. Next, these three classes of siRNA were tested for their ability to act during HDV genome replication. It was found that only siRNA targeted against HDV mRNA sequences could interfere with HDV genome replication. In contrast, siRNA targeted against genomic and antigenomic RNA sequences had no detectable effect on the accumulation of these RNAs. Reconstruction experiments with nonreplicating HDV RNA sequences support the interpretation that neither the potential for intramolecular rod-like RNA folding nor the presence of the delta protein conferred resistance to siRNA. In terms of replicating HDV RNAs, it is considered more likely that the genomic and antigenomic RNAs are resistant because their location within the nucleus makes them inaccessible to siRNA-mediated degradation.

Animals↗
PubMed1991

Identification of a new viroid as the putative causal agent of pear blister canker disease.

A small RNA species with the structural and functional properties characteristic of viroids has been isolated from three different pear sources each of which induced symptoms of the pear blister canker (PBC) disease when indexed in the pear indicator A 20. A close association between this RNA and PBC disease was established, since two of the three studied sources were known to be affected by this malady only, and the viroid was not detected in healthy pear tissue. Moreover, the PBC-associated viroid (PBCVd) replicated when purified preparations were inoculated into cucumber and pear plants. PBCVd behaved in denaturing polyacrylamide gels as a circular RNA with a molecular size of approximately 315 nucleotide residues. Analysis by dot blot hybridization indicated that PBCVd shares similarities in sequence with peach latent mosaic viroid and hop stunt viroid, and to a lesser extent with apple scar skin viroid.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

RNA structural features responsible for potato spindle tuber viroid pathogenicity.

The native structure of potato spindle tuber viroid (PSTVd) contains a series of short double helices and small internal loops that are organized into five structural domains. Nucleotides within the pathogenicity domain are known to play a critical role in modulating PSTVd symptom expression, and it has been suggested that disruption of a comparatively unstable "premelting region" within the pathogenicity domain may be required for disease induction. We have used a combination of quantitative bioassays, temperature gradient gel electrophoresis of circularized RNA transcripts, and thermodynamic calculations to compare the biological and structural properties of 12 representative PSTVd sequence variants. Certain mutations appeared to act indirectly, downregulating pathogenicity by suppressing the rate of PSTVd replication/accumulation. The effects of other mutations appeared to be more direct, but there was no consistent correlation between symptom severity and melting temperature. Taking into account the three-dimensional shape of RNA helices, comparison of the optimal secondary structures for these variants point to major differences in the geometry of their pathogenicity domains; i.e., variants producing intermediate symptoms possess a linear arrangement of three consecutive helices, whereas for variants producing mild or severe symptoms this domain is bent in opposing directions. Such alterations in RNA structure together with concomitant alterations in RNA-protein interaction(s) may be the primary cause of viroid pathogenicity.

Base Sequence↗
PubMed2004

Biolistic inoculation of plants with viroid nucleic acids.

Parameters for biolistic transfer of viroid nucleic acids using a Helios Gene Gun device were assayed. The main achievement of this method is high efficiency of inoculation with linear monomeric viroid cDNAs and RNAs. This greatly facilitates the study of mutated sequence variants, viroid libraries and mixed populations. The lower limits for efficient inoculation of monomeric cDNA fragments with the sequence of potato spindle tuber viroid (PSTVd) and native PSTVd RNA as detected 21 days p.i. are in the range of 50 ng and 200 pg per tomato plant, respectively. At a higher dose, i.e. 2 ng of native RNA per plant, biolistic transfer causes drastic stunting compared to conventional mechanical inoculation, which points to higher PSTVd titers after the biolistic transfer. Infection is readily achieved with exact length monomeric RNA transcripts having 5'-triphosphate and 3'-OH termini in amounts ranging from 2 to 20 ng per plant, suggesting no need for any supplementary modifications of ends or RNA circularization. The biolistic transfer is efficient for viroid "thermomutants", which exhibit low or no infectivity with conventional mechanical inoculation with Carborundum. The biolistic inoculation is also efficient for two other members of the Pospiviroidae family, hop stunt and hop latent viroid.

Biolistics↗
PubMed2026

Context-dependent roles of DHX9 in Cancer: Molecular mechanisms, biomarker potential, and therapeutic perspectives.

DExH-box helicase 9 (DHX9) is a multifunctional nucleic acid helicase that participates in R-loop homeostasis, genome maintenance, RNA metabolism, and innate immune signaling. Accumulating evidence has linked aberrant DHX9 expression or activity to tumorigenesis, tumor progression, treatment response, and patient prognosis. However, its role in cancer is highly context-dependent, rather than uniformly oncogenic or tumor suppressive. Depending on its molecular partners, subcellular localization, post-translational modifications, tumor genotype, and immune microenvironment, DHX9 may either promote malignant phenotypes or contribute to tumor-restraining processes. In this review, we summarize the molecular characteristics and regulatory properties of DHX9, discuss its roles in genome stability, transcriptional and post-transcriptional control, circular RNA (circRNA) biogenesis, and tumor-immune crosstalk, and evaluate its emerging value as a potential biomarker and therapeutic target. We also highlight key challenges in this field, including mechanistic heterogeneity, insufficient translational validation, and the urgent need for context-informed patient stratification to maximize the clinical utility of DHX9-targeted strategies.

Humans↗
PubMed1994

Structural characteristics of the stable RNA introns of archaeal hyperthermophiles and their splicing junctions.

Five pre-23 S rRNA introns have been characterized for hyperthermophilic archaea. The small ones constitute stable core structures while the three larger ones contain, in addition, open reading frames, two of which encode homing-type endonucleases. The higher order structures of in vitro transcripts of the introns and their exon-intron junctions were examined using chemical and ribonuclease probes specific for unpaired nucleotides, double helical regions and helix-loop junctions. The experimental data support both the formation of a "bulge-helix-bulge" structural motif at the exon-intron junctions, which is recognized by a cleavage enzyme, and the presence of an adjacent core structure consisting of a long, stable, stem-loop structure. Moreover, the data were used to derive secondary structural models for the three large introns which form stable, circular, RNA species, in vivo. Each exhibits extensive secondary structure and additional, as yet undefined, higher order structure, compatible with their high level of stability in vivo; limited structural similarities were detected between the two introns encoding the homing-type endonucleases. The data are compatible with the hypothesis that RNA introns can be transmitted between archaeal hyperthermophile cells.

Archaea↗
PubMed2003

Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA.

Elements of local tertiary structure in RNA molecules are important in understanding structure-function relationships. The loop E motif, first identified in several eukaryotic RNAs at functional sites which share an exceptional propensity for UV crosslinking between specific bases, was subsequently shown to have a characteristic tertiary structure. Common sequences and secondary structures have allowed other examples of the E-loop motif to be recognized in a number of RNAs at sites of protein binding or other biological function. We would like to know if more elements of local tertiary structure, in addition to the E-loop, can be identified by such common features. The highly structured circular RNA genome of the hepatitis D virus (HDV) provides an ideal test molecule because it has extensive internal structure, a UV-crosslinkable tertiary element, and specific sites for functional interactions with proteins including host PKR. We have now found a UV-crosslinkable element of local tertiary structure in antigenomic HDV RNA which, although differing from the E-loop, has a very similar pattern of sequence and secondary structure to the UV-crosslinkable element found in the genomic strand. Despite the fact that the two structures map close to one another, the sequences comprising them are not the templates for each other. Instead, the template regions for each element are additional sites for potential higher order structure on their respective complementary strands. This wealth of recurring sequences interspersed with base-paired stems provides a context to examine other RNA species for such features and their correlations with biological function.

Binding Sites↗
PubMed2002

Activation of a p44 pseudogene in Anaplasma phagocytophila by bacterial RNA splicing: a novel mechanism for post-transcriptional regulation of a multigene family encoding immunodominant major outer membrane proteins.

Immunodominant 44 kDa major outer membrane proteins of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) are encoded by the p44 multigene family. One of the paralogues, p44-18 is predominantly expressed by A. phagocytophila in mammalian hosts, but is downregulated in the arthropod vector. The expression of p44-18 was upregulated in A. phagocytophila cultivated in HL-60 cells at 37 degrees C compared with 24 degrees C. However, the molecular mechanism of such gene expression was unclear, as p44-18 has a pseudogene-like structure, i.e. it lacks an AUG start codon and is out of frame with an upstream overlapping paralogue, p44-1. In the present study, we found that an amplicon detected by reverse transciption-polymerase chain reaction (RT-PCR) [808 basepair (bp)] for the p44-1/p44-18 gene locus was smaller than that detected by PCR with the genomic DNA (1652 bp) in the A. phagocytophila-infected HL-60 cells cultured at 37 degrees C. A circularized RNA molecule corresponding to the 844 bp region missing from the locus in the RT-PCR product was detected by inverse RT-PCR, indicating that this is an intron (designated p44-1 intervening sequence, p44-1 IVS). The splicing event of p44-1 IVS was also observed when the p44-1 IVS-carrying plasmid was introduced into Escherichia coli, suggesting that the splicing is sequence-dependent. Structural analysis and in vitro splicing experiments of p44-1 IVS suggested that this is likely to represent a new class of introns in eubacteria. The primer extension analysis showed the presence of a putative sigma(32)-type promoter in region upstream from p44-1. Collectively, the novel RNA splicing and the temperature-dependent transcription may account for the dominant p44-18 expression in mammals.

Amino Acid Sequence↗
PubMed2003

Translational control by the 3'-UTR: the ends specify the means.

In most cases, translational control mechanisms result from the interaction of RNA-binding proteins with 5'- or 3'-untranslated regions (UTRs) of mRNA. In organisms ranging from viruses to humans, protein-mediated interactions between transcript termini result in the formation of an RNA loop. Such RNA 'circularization' is thought to increase translational efficiency and, in addition, permits regulation by novel mechanisms, particularly 3'-UTR-mediated translational control. Two general mechanisms of translational inhibition by 3'-UTR-binding proteins have been proposed, one in which mRNA closure is disrupted and another in which mRNA closure is required. Experimental evidence for the latter is provided by studies of interferon-gamma-mediated translational silencing of ceruloplasmin expression in monocytic cells. A multi-species analysis has shown that, in most vertebrates, 3'-UTRs are substantially longer than their 5' counterparts, indicating a significant potential for regulation. In addition, the average length of 3'-UTR sequences has increased during evolution, suggesting that their utilization might contribute to organism complexity.

3' Untranslated Regions↗