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PubMed2026

Cold-adapted RNA polymerase from Pseudomonas phage Njord improves synthesis of therapeutic mRNA.

An RNA polymerase identified in the genome of Pseudomonas phage Njord offers a promising tool for the synthesis of mRNA and other therapeutic nucleic acids. Originating from a marine microbial ecosystem, Njord RNAP transcribes RNA at high yield even under low temperature conditions. Key properties of the enzyme relevant to mRNA synthesis are presented including transcriptional fidelity, promoter specificity, incorporation of modified nucleotides, and the impurity profile of the RNA. Specific attention is given to the formation of contaminating double-stranded RNA (dsRNA) species. Analysis of transcription reactions shows that DNA-templated promoter-independent transcription is a major source of detectable dsRNA impurities and that Njord RNAP displays a minimal level of this activity. Consistent with the known inflammatory role of dsRNA in synthetic mRNA, transcriptomic analysis of cell culture and a live animal study demonstrates that mRNA synthesized with Njord RNAP elicits only a minimal immune response. This natural enzyme enables efficient mRNA synthesis at ambient temperature and produces transcripts essentially free of dsRNA, offering significant potential to streamline mRNA manufacturing processes.

DNA-Directed RNA Polymerases↗
PubMed2026

Comparative evaluation of chitosan-based and star polycation nanocarriers for enhanced RNAi efficacy targeting CmFibL in Cnaphalocrocis medinalis.

BACKGROUND: The rice leaf folder, Cnaphalocrocis medinalis, causes substantial rice yield losses through larval leaf-rolling behavior. RNA interference (RNAi) offers a sustainable alternative, but its application in Lepidoptera is hindered by dsRNA degradation and poor cellular uptake. This study developed nanocarrier-mediated dsRNA delivery to overcome these limitations. RESULTS: Three nanocarriers - chitosan (CS), chitosan-tripolyphosphate (CS-TPP), and star polycation (SPc) - were compared for enhancing RNAi efficiency targeting the C. medinalis fibroin light chain gene (CmFibL). CS-TPP and SPc achieved 61% and 55% silencing efficiency, respectively, representing 2.7-fold improvement over naked dsRNA (23%). All nanocarriers protected dsRNA from RNase A (30 min) and midgut fluid (6 h) degradation. CmFibL knockdown caused severe silk defects, prolonged pupal duration by 23%, reduced pupal weight by 33%, and decreased leaf-rolling damage by 31% in glasshouse cage trials. Transcriptomics revealed down-regulation of amino acid metabolism and activation of endoplasmic reticulum (ER) stress and immune responses. No off-target effects were detected in human genome, nor in any predators or parasitoids sharing the same ecological niche. CONCLUSION: CS-TPP and SPc nanocarriers effectively enhance RNAi efficiency in a Lepidopteran pest. Targeting CmFibL disrupts silk-mediated feeding shelters with minimal ecological risk, providing a practical framework for field application of RNAi-based biopesticides against leaf-rolling rice pests. © 2026 Society of Chemical Industry.

Animals↗
PubMed1979

The regulation of RNA synthesis in yeast IV. Synthesis of double-stranded RNA.

A study has been made of the regulation of the synthesis of Pl double-stranded (ds) RNA, the genome of the yeast virus-like particle. When yeast protein synthesis is prevented by starvation for a required amino acid or by addition of cycloheximide, the rate of Pl dsRNA synthesis is reduced markedly. During nitrogen starvation the synthesis of Pl dsRNA persists but is accompanied by the degradation of pre-existing molecules. This degradation appears to require the induction of new enzymes and it is likely that the breakdown products are used to enable the cell to complete its division cycle. However, all of the copies of the VLP genome are not degraded in this process, some are conserved and can replenish the amount of Pl dsRNA on return to growth conditions. The controls which must operate on Pl dsRNA synthesis are discussed and compared with those exerted on nuclear RNA synthesis in yeast.

Cell Division↗
PubMed1979

Heterogeneous nuclear RNA double-stranded regions probed in living HeLa cells by crosslinking with the psoralen derivative aminomethyltrioxsalen.

The psoralen derivative aminomethyltrioxsalen (AMT, 4'-aminomethyl-4,5',8-trimethylpsoralen) has been employed as a probe for heterogeneous nuclear RNA (hnRNA) double-stranded regions in experiments with living HeLa cells. hnRNA ribonucleoprotein (hnRNP) particles were purified from untreated or AMT-treated cells after irradiation with 365-nm light, and double-stranded hnRNA regions (dsRNA) were isolated by RNase A + T1 digestion of hnRNP, followed by preparative Cs2SO4 isopycnic centrifugation. The purified, hnRNP-derived dsRNA was then assayed for interstrand crosslinks by measurement of its "snapback" to RNase-resistant form after thermal denaturation. By this procedure, the amount of crosslinked dsRNA was found to be increased 3- to 7-fold in cells exposed to AMT in vivo. The levels of crosslinking in vivo compared favorably with those observed in model experiments with pure dsRNA in vitro. These results establish that double-stranded hnRNA regions exist in the living cell, and they further demonstrate that these base-paired regions are organized as rather accessible sites within the nucleus.

Furocoumarins↗
PubMed1976

A compact form of double-stranded RNA in solutions containing poly(ethyleneglycol).

Molecules of single-stranded ribosomal RNA and double-stranded replicative form of phage f2 RNA (dsRNA) adopt a compact form in solutions, containing sufficiently high concentrations of salt (NaCl) and polymer (PEG). However, only in the cases of native dsRNA molecules the compact particles are characterized by a regular internal structure, which accounts for the appearance of an intense positive band in CD spectra. Heating or acidification of PEG-containing solutions of dsRNA leads to the disappearance of the intense positive CD band, which results from the "destruction" of the regular internal structure of compact particles. Comparison of properties of DNA and dsRNA compact particles formed in PEG-containing water-salt solutions suggests the existence of similar mechanisms of compactization of double-stranded polynucleotides.

Circular Dichroism↗
PubMed1979

Evidence for secondary structure in poliovirus virion RNA demonstrated by antibodies against double-stranded RNA.

Poliovirus particle RNA has been considered to have little secondary structure. Specific binding of poliovirion RNA by antibodies against double-stranded RNA (dsRNA) has been confirmed and further characterized by a radioimmunoassay using Staphylococcus aureus protein A to precipitate the nucleic acid-antibody complex. Competitive binding studies between virion single-stranded RNA (ssRNA) and poly(I).poly(C) demonstrated that the dsRNA effectively inhibited binding of radiolabelled poliovirion RNA by the anti-dsRNA antibodies but the virion RNA was a poor competitor of radiolabelled ds RNA. This indicates that both RNAs reacted with the same species of antibodies in the sera, but avidity of the antibodies for dsRNA was greater than for the poliovirion RNA.

Animals↗
PubMed2021

Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake.

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Ascomycota↗
PubMed2026

Endosymbiotic theory of aging revisited: Age-related leakage of mitochondrial dsDNA/RNA stimulates cytosolic nucleic acid sensors which remodel the immune network and promote the aging process.

About 1.5-2 billion years ago, an endosymbiosis between aerobic α-proteobacteria and anaerobic archaeal cells generated mitochondria, i.e., organelles capable of producing oxidative energy. The bacterial genome was fundamentally reduced and a circular mitochondrial genome evolved containing mainly the genes coding for the subunits of the electron transport chain. Before the symbiotic event, there existed a virus-host co-evolution which involved the development of sensors for detecting dangerous viral DNA/RNA molecules. Endosymbiosis supplied eukaryotic cells not only with an oxidative powerhouse to allow the evolution of more complex multicellular organisms but it also meant that cells now housed an organelle which was able to generate reactive oxygen species (ROS) and to leak mitochondrial DNA (mtDNA) and double-stranded RNA (dsRNA) into the cytoplasm. There is now abundant evidence that during aging and age-related diseases mitochondria are prone to release both mtDNA and dsRNA. In the cytoplasm, mtDNA/dsRNA molecules activate a number of cytosolic nucleic acid sensors leading to the secretion of type-1 interferons (IFN) and many other cytokines which promote an age-related proinflammatory state. Currently, it is known that mtDNA can activate the cGAS-STING pathway, AIM2 inflammasomes, IFI16 receptors, and ZBP1 sensors and in addition mitochondrial dsRNA stimulates RIG-1/MDA5 signaling. Interestingly, there is abundant evidence that all these receptors are drivers of cellular senescence and inflammaging. For decades, there has been mounting evidence that mitochondria have a crucial role in the aging process. We will examine this question from the perspective of evolution and propose that mitochondrial evolution created an endogenic source for the leakage of dangerous mtDNA/dsRNA which subsequently stimulated cytosolic DNA/RNA sensors, an evolutionarily conserved viral defence mechanism. It seems that these two evolutionary events provided not only the basis for the inevitable process of aging but also ensuring the death of parental organisms.

Aging↗
PubMed2026

Viral community in Aspergillus spp. isolated from commercially available fermented dried bonito.

Katsuobushi is a traditional processed seafood product used in Japanese-style cooking, and when it is produced through fermentation by fungi, it is called karebushi. The fungi involved in katsuobushi fermentation are collectively referred to as katsuobushi molds. We previously discovered seven novel viruses from katsuobushi molds and determined their genome sequences. However, our previous explorations used only nine fungal strains available from culture collections, leaving the diversity of viruses infecting fungi involved in katsuobushi fermentation unclear. Therefore, in this study, we aimed to isolate fungi from commercially available karebushi and clarify the prevalence of viruses in the isolates. Karebushi produced by three manufacturers was obtained, and 30 fungal strains (including Aspergillus spp.) were isolated from each. Double-stranded RNA (dsRNA) fractions were prepared from the mycelia of the isolated strains. Electrophoresis suggested that a relatively high proportion of the isolates harbored dsRNA elements consistent with RNA virus infection (30-70% per manufacturer; 59% overall). Furthermore, dsRNA sequencing identified four novel viruses in isolates of Aspergillus chevalieri and Aspergillus montevidensis: a beny-like virus, a gammapartitivirus, a narnavirus, and a victorivirus, in addition to two previously reported viruses. Notably, this represents the first report of a beny-like virus in Aspergillus spp. This study provides insights into the diversity of viruses infecting fungi involved in katsuobushi fermentation.

Aspergillus↗
PubMed2022

ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

Humans↗
PubMed1975

Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA and oxidized glutathione: indirect mode of action on polypeptide chain initiation.

In the presence of added double-stranded RNA or oxidized glutathione, protein synthesis in heminsupplemented reticulocyte lysates declines abruptly after 8-12 min of incubation at 30 degrees. The kinetics of amino-acid incorporation are very similar to those seen when lysates incorporation are very similar to those seen when lysates are incubated in the absence of added hemin. The inhibitory effects of double-stranded RNA (dsRNA) and oxidized glutathione (GSSG) are partially overcome by a homogeneous initiation factor, IF-MP, which also stimulates protein synthesis in hemin-deficient lysates. This factor is involved in the binding of Met-tRNAfmet to 40S ribosomal subunits during protein chain initiation. However, neither dsRNA alone nor GSSG alone significantly inhibits formation of [40S subunit-Met-tRNAf] complexes induced in reticulocyte lysates by dsRNA or GSSG involves one or more components present in the lysates but absent from the fractionated in vitro system. Such components may be related to the translational inhibitor that is active in hemin-deficient lysates.

Animals↗
PubMed1975

Ionic strength effects on the stability and conformation of Penicillium chrysogenum mycophage double stranded RNA.

The effect of [Na+] on the stability and conformation of penicillium chrysogenum mycophage dsRNA (PCMdsRNA) was investigated using CD and UV optical techniques. Thermal melting profiles reveal prominent fine structure attributed to at least four regions of structural dissimilarity. A constant increased thermal stability of the dsRNA compared to DNA of the same base composition was observed over a concentration range of 1.5 times 10- minus 4 M to 4.5 times 10- minus 2 M Na+. At low ionic strengths ([Na+] less than 10- minus 3 M) Tm becomes independent of further decrease in [Na+] unless the dsRNA is exposed to high concentrations of EDTA, suggesting the involvement to tightly bound divalent cation. At relatively high ionic strengths ([Na+] greater than 0.1 M) a postulated A leads to A' ... conformation change occurs.

Circular Dichroism↗
PubMed1977

Induction of yeast killer factor mutations.

Two related killer strains of Saccharomyces cerevisiae were mutagenized and screened for nonkiller variants. About 20% of the mutants derived from one strain lacked all detectable double-straned ribonucleic acid (dsRNA). About 70% of the mutants from the other strain lacked one of the dsRNA species normally associated with the killer factor and had in its place another species of dsRNA with a lower molecular weight.

Ethyl Methanesulfonate↗
PubMed1975

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.

Adenine Nucleotides↗
PubMedDate not supplied

[Circular dichroism of DNA complexes with dyes. III. Effect of latent optical activity and the structure of the complexes].

Circular dichroism anisotropy was studied both theoretically and experimentally for the complexes of DNA and dsRNA with dyes (proflavine, 2,7-di-t-butyl proflavine, "Hoechst-33258") and antibiotics (distamycin A, netropsin and olivomycin). Theoretical analysis showed that general features of CD anisotropy, revealed in the previous studies (CD components--delta epsilon parallel to and delta epsilon perpendicular--are ten times or more bigger than the CD-effect without orientation, and delta epsilon parallel to approximately 2 delta epsilon perpendicular) are due to the existence of a specific effect named "latent" optical activity (LOA). This effect can be observed in many cases of non-chiral symmetrical chromophores if they are oriented. The effect is due to the excitation of an electrical dipole transition and a perpendicular magnetic dipole transition (or quadrupole transition) of a molecule. The amplitude and the sign of the LOA-effect depends on the orientation of the chromophores with respect to the light beam; with a random orientation the mutual compensation of LOA-effects of different chromophores happens and no LOA-effect appears. The analitycal expressions relating the value of LOA-effect of the system with electronical characteristics of the chromophores and the geometrical parametra of their arrangement was obtained. The experimental data obtained for the oriented complexes of DNA and dsRNA with proflavine made it possible to determine an angle between the chromophore and the plane perpendicular to the DNA axis--gamma. For the calf thymus DNA gamma = = + 1.8 +/- 0.4 degrees, for the phage T2 DNA gamma = + 2.2 +/- 0.4 degrees, and for phage f2 dsRNA gamma=--3.5 +/- +/- 0.5 degrees. These results, obtained at relatively low concentrations of the bound proflavine (r approximately 0.01), are in accordance with the intercalating mode of the dye binding. A study of CD anisotropy of DNA complexes with other ligands showed that many different chromophores possess LOA-effect. This phenomenon can be used to obtain both spectroscopic and structural information about the systems similar to those reported here.

Acridines↗
PubMed1978

Cleavage of pre-mRNA sequences by ribonucleases bound to nuclear RNP particles of rat liver.

The 30S nuclear RNP particles from rat liver have been shown to split the double-stranded- (ds) and single-stranded (ss) sequences of nuclear pre-mRNA. Experiments performed in vitro have demonstrated that 1) a 5'-exonuclease and an endonuclease specific for double-stranded pre-mRNA sequences exist in the 30S pre-mRNP particles; 2) in dsRNA monophosphorylated 5'-termini arose in the course of incubation with 30S RNP and most of the products remained double-stranded. The analysis of terminal pNp nucleotides revealed a relatively high ratio of pPyp in the cleaved dsRNA, whereas the nucleosides in 5'-terminal pNp of ssRNA showed nearly random distribution. Our results provide a possible explanation for the appearance of pNp termini during the processing of nuclear pre-mRNA of mammalian cells.

Animals↗
PubMed1977

Increased synthesis of abundant poly(A)-containing RNA in a DNA defective mutant of Saccharomyces cerevisiae containing the "killer character".

A Saccharomyces cerevisiae strain which contains both the "killer character" and a ts mutation in the initiation of nuclear DNA synthesis (cdc4) was studied. Incubation of this strain at the restrictive temperature caused a 3--4 fold increase in the relative rate of synthesis of abundant RNA which contains poly(A) and a 2--3-fold increase in the relative rate of synthesis of killer dsRNA. Thus, the amount of killer dsRNA found in these cells seems to be correlated to the amount of abundant poly(A)-RNA.

DNA↗
PubMed2025

Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.

Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.

RNA Editing↗