Improved separation of glyoxalated viroid RNAs and other small nucleic acids in polyacrylamide gels containing urea.
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Biotechnology↗
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Molecular biologic and pathogenetic analysis of hepatitis delta virus.
Hepatitis delta virus (HDV) consists of an envelope derived from hepatitis B virus (HBV) and a nucleocapsid consisting of hepatitis delta antigen and a circular RNA genome. Both the delta antigen and the RNA possess many unique properties which play crucial roles in the life cycle of HDV. They may also contribute indirectly to the mechanism of viral pathogenesis.
Antigens, Viral↗
Evidence for a model ancestral viroid.
The generation of a phylogenetic tree of viroids and viroid-like plant satellite RNAs via computer analysis, coupled with several conspicuous biochemical characteristics of the rolling circle replication of these RNAs -including both self-cleavage and self-ligation- leads us to propose the peach latent mosaic viroid (PLMVd) as a current "living fossil" dating from a precellular world. Incorporated within this proposal is a revised mechanism of PLMVd rolling circle replication which requires a minimal protein involvement.
Evolution, Molecular↗
Oligoribonucleotide circularization by 'template-mediated' ligation with T4 RNA ligase: synthesis of circular hammerhead ribozymes.
Circular hammerhead ribozymes were synthesized from linear oligoribonucleotides using T4 RNA ligase. Some of the precursors could not be efficiently circularized under standard conditions. For these molecules, the use of a DNA template allowed their efficient circularization. The template was designed to prevent the precursor from folding into an unsuitable structure. The template allowed circular ribozymes as small as 15 nucleotides in length to be efficiently synthesized at concentrations as high as 50 microM in the ligation reaction. The circular products retained their biological activity.
Bacteriophage T4↗
Binding of ethidium bromide to ribosomal RNA. Absorption, fluorescence, circular and electric dichroism study.
The interaction between ethidium bromide and ribosomal RNA has been studied by means of absorption, fluorescence, circular and electric dichroism measurements in the near ultraviolet and visible regions at low ionic strength (1 . 10(-3) and 6 . 10(-3). The results have been interpreted on the basis of a model of interaction involving the intercalation of the phenanthridinium ring of the dye in the double-stranded regions of the RNA molecule, resulting in an increase of the dye-dye interactions as compared to DNA, and a stiffening of the intercalation regions.
Binding Sites↗
Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract.
Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed.
Cell-Free System↗
Circularization of retroviral genomic RNA and the control of RNA translation, packaging and reverse transcription.
Translation, packaging and reverse transcription of the genomic RNA of retroviruses appear to be regulated by short and long range RNA-RNA interactions which take place within the 5'-600 nt and between the 5' and 3' untranslated sequences. The 5' (R and U5) and 3' (Dr and U3) domains of the genomic RNA together with the nucleic acid binding protein (NBP) would control the balance between the open state of the viral genomic RNA, correlated with an efficient RNA translation and the closed state, with the circular viral RNA efficiently packaged into virions. Retroviral NBP might well drive the packaging of the viral RNA as well as improve reverse transcription of the circular virion RNA.
Amino Acid Sequence↗
Poliovirus RNA replication requires genome circularization through a protein-protein bridge.
The mechanisms and factors involved in the replication of positive stranded RNA viruses are still unclear. Using poliovirus as a model, we show that a long-range interaction between ribonucleoprotein (RNP) complexes formed at the ends of the viral genome is necessary for RNA replication. Initiation of negative strand RNA synthesis requires a 3' poly(A) tail. Strikingly, it also requires a cloverleaf-like RNA structure located at the other end of the genome. An RNP complex formed around the 5' cloverleaf RNA structure interacts with the poly(A) binding protein bound to the 3' poly(A) tail, thus linking the ends of the viral RNA and effectively circularizing it. Formation of this circular RNP complex is required for initiation of negative strand RNA synthesis. RNA circularization may be a general replication mechanism for positive stranded RNA viruses.
Base Sequence↗
Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.
The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.
Arenaviridae↗
Tm studies of a tertiary structure from the human hepatitis delta agent which functions in vitro as a ribozyme control element.
Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; > 50% of the complexes remain following incubation in 1 mM EDTA at 60 degrees C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures approximately 10 degrees C lower in buffers containing 0.5 mM MgCl2 and 100 mM NaCl than in buffers containing 0.5 mM MgCl2 with no NaCl (78.1 compared with 87.7 degrees C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.
Base Sequence↗
Pathway modulation, circular permutation and rapid RNA folding under kinetic control.
The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity. The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures. To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken. This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC. While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct. Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions. This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA. However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms. These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control.
Bacillus subtilis↗
Detecting known neoepitopes, gene fusions, transposable elements, and circular RNAs in cell-free RNA.
MOTIVATION: Cancer is the second leading cause of death worldwide, and although there have been advances in treatments, including immunotherapies, these often require biopsies which can be costly and invasive to obtain. Due to lack of pre-emptive cancer detection methods, many cases of cancer are detected at a late stage when the definitive symptoms appear. Plasma samples are relatively easy to obtain, and they can be used to monitor the molecular signatures of ongoing processes in the body. Profiling cell-free DNA is a popular method for monitoring cancer, but only a few studies have explored the use of cell-free RNA (cfRNA), which shows the recent footprint of systemic transcription. RESULTS: Here, we developed FastNeo, a computational method for detecting known neoepitopes in human cfRNA. We show that neoepitopes and other biomarkers detected in cfRNA can discern Hepatocellular carcinoma patients from the healthy patients with a sensitivity of 0.84 and a specificity of 0.79. For colorectal cancer we achieve a sensitivity of 0.87 and a specificity of 0.8. An important advantage of our cfRNA based approach is that it also reports putative neoepitopes which are important for therapeutic purposes. AVAILABILITY AND IMPLEMENTATION: The FastNeo package is available at https://github.com/yashumayank/FastNeo and https://zenodo.org/records/11521368. The benchmark pipelines to detect Immune Epitope database and Tumor-Specific Neoantigen database neoepitopes using HaplotypeCaller, bcftools, and Lofreq, and to run FastNeo with STAR instead of Bowtie2 are also available in the above github repository.
Humans↗
Production of circular form of streptavidin RNA aptamer in vitro.
RNA aptamer is one of functional RNAs selected experimentally from randomized RNA pools, and has a high affinity and specificity against the target such as a protein and a small ligand. For therapeutic use of RNA aptamer in vivo, both no side effects and escaping from RNase-induced degradation may be required. Here we show a first evidence of producing a circular streptavidin aptamer in vitro generated by self-splicing permutated intron-exon (PIE) sequences derived from gene td of T4 bacteriophage. In spite of its complex higher-order structure, the aptamer sequence was circularized. The circularized aptamer retains its binding affinity toward streptavidin and its binding was inhibited in the presence of biotin. The availability of circularizing the RNA aptamer in vitro would be a candidate for a new methodology for RNA therapeutics.
Aptamers, Nucleotide↗
A 5' RNA element promotes dengue virus RNA synthesis on a circular genome.
The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.
Dengue Virus↗
Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction.
In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.
Chemical Fractionation↗
Specific site of action for single-strand specific nuclease on the double-stranded circular DNA intermediates of an avian RNA tumor virus.
Circular viral DNA intermediates obtained from the quail tumor line, QT6, at 1 day after infection, were opened at one specific location by the single-strand specific nuclease, S1, of Aspergillus oryzae. This site was no longer accessible to the S1 nuclease when circles were first opened at another location with a restriction endonuclease.
Alpharetrovirus↗
Detecting and quantifying circular RNAs in terabyte-scale RNA-seq datasets with CIRI3.
To address recent challenges in circular RNA (circRNA) analysis, we present CIRI3, a tool for circRNA detection and quantification in terabyte-scale RNA-sequencing datasets. Using dynamic multithreaded task partitioning and a blocking search strategy for junction reads, CIRI3 is an order of magnitude faster than existing tools, while providing increased accuracy. We identified differentially spliced circRNAs across 2,535 cancer-related samples, and constructed a pretraining model and a biomarker network provided as the CIRIonco database.
RNA, Circular↗
Generation and characterization of circular Bacillus subtilis RNase P RNA; activation by RNase P protein.
A circular form of Bacillus subtilis ribonuclease P RNA (C-P RNA) was generated in vitro by splicing permuted intron-exon (PIE) sequences containing the P RNA sequence. Steady-state cleavage of pre-tRNA(Asp) catalyzed by circular P RNA is slightly faster than the linear form. Furthermore, steady-state turnover catalyzed by circular RNase P RNA is activated by the addition of the Bacillus subtilis protein component of RNase P, to a rate constant equal to the linear holoenzyme under identical conditions. Also, the circles are resistant to nuclease degradation, have less sequence heterogeneity, and may enhance the formation of a unique structure. Therefore, circular forms of RNase P RNA should prove useful for mutagenesis and structural studies.
Bacillus subtilis↗