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[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↗
PubMed1978

Electron microscopic heteroduplex analysis of "killer" double-stranded RNA species from yeast.

Wild-type and mutant double-stranded RNA (dsRNA) species from the yeast Saccharomyces cerevisiae were studied by electron microscopic heteroduplex mapping to determine the sequence relationships among the different RNA molecules. Three mutant dsRNAs, 1.5, 1.4, and 0.73 kilobase, were found to be derived by the same internal deletion of the wild-type (I83 kilobases) molecule. This deletion includes a wild-type (1.83 kilobases) molecule. This deletion includes a segment of about 200 base pairs that was estimated to be nearly 100% A+U. In addition, the sequences of the two larger mutant RNA species are tandem, direct duplications. One of the duplicated molecules appears to have a second internal deletion that occurred after the duplication. The mutant dsRNAs are functionally similar to the defective interfering virus particles of animal viruses--all of the mutant species prevent the propagation of the wild-type dsRNA when both are present in the same cell. The four dsRNAs share the same sequences at their termini, a finding that may suggest that these sequences are important for the replication of the dsRNAs.

Base Sequence↗
PubMed1979

In situ phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase.

Protein synthesis initiation in reticulocyte lysates is inhibited by heme deficiency, low levels of double-stranded RNA (dsRNA), oxidized glutathione (GSSG), or the purified kinase (HRI) that acts on the alpha polypeptide of eukaryotic initiation factor 2 (eIF-2alpha). The phosphoprotein profiles produced in lysates in response to these various conditions have been monitored directly in lysates after labeling for brief periods with pulses of [gamma-(32)P]ATP. The [(32)P]phosphoprotein profiles were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide slab gels under conditions in which the HRI and eIF-2alpha polypeptides were clearly distinguished. All four modes of inhibition produced a rapid phosphorylation of eIF-2alpha compared to control lysates, which displayed little or no phosphorylation of eIF-2alpha. In heme-deficient lysates, phosphorylation of eIF-2alpha occurred rapidly both before and after the shut-off of protein synthesis; the delayed addition of hemin to these lysates resulted in a decrease in the phosphorylation of eIF-2alpha and the subsequent restoration of protein synthesis. These data suggest that rapid turnover of phosphate occurs at the site(s) of eIF-2alpha phosphorylation. In lysates inhibited by heme deficiency, GSSG, or added HRI, the phosphorylation of eIF-2alpha was accompanied by the rapid in situ phosphorylation of HRI. The inhibition of initiation induced by dsRNA was accompanied by the phosphorylation of eIF-2alpha and a 67,000-dalton polypeptide but not HRI. These observations in situ indicate that (i) the phosphorylation of eIF-2alpha is the critical event in these inhibitions of protein chain initiation, and (ii) the phosphorylation of HRI is associated with its activation in heme deficiency.

Adenosine Triphosphate↗
PubMed1976

Size and structure of the genome of infectious pancreatic necrosis virus.

The genome of infectious pancreatic necrosis virus consists of two segments of dsRNA, in equimolar amounts, with molecular weights of 2.5 X 10(6) and 2.3 X 10(6) daltons, as determined by polyacrylamide gel electrophoresis and autoradiography. The viral RNA was resistant to ribonuclease, and in sucrose gradient it co-sedimented at 14S with RNase resistant RNA from virus infected cells. Upon denaturation in 98% formamide, the viral genome sedi-mented at 24S in formamide sucrose gradient and became sensitive to RNase. Denatured 24S viral RNA did revert to its undenatured 14S form upon recentrifugation in aquaeous sucrose gradient (0.1 M NaCL), but co-sedimented with the denatured large size class of reovirus 25S RNA. The same results were obtained if the native viral RNA was pre-treated with ribonuclease before denaturation, indicating the absence of exposed single strainded regions in the viral genome. Since infectious pancreatic necrosis virus contains only two dsRNA segments it does not belong to the family Reoviridae and may represent a new group of viruses.

Animals↗
PubMed2025

Detection of Orsay viral replication intermediates reveals spatial and regulatory links to Caenorhabditis elegans innate immune responses.

For a positive-strand RNA virus, the encoded viral RNA-dependent RNA polymerase (oRdRP) synthesizes complementary antigenome strand and uses it as a template for amplifying the viral genome, generating various replication intermediates. Structural proteins and viral genome are packaged into virions, but the fate of replication intermediates is underexplored. Here, we investigate Orsay Virus (OV) replication intermediates, including antigenome, oRdRP and double stranded RNA (dsRNA), using PCR and fluorescence-based imaging in C. elegans intestines. As for other positive-strand RNA viruses, we find that genome is in vast excess of antigenome. Antigenome is only visualized in cells when using denaturation protocols, indicating basepaired intermediates. OV antigenome is observed with distinct cytoplasmic and perinuclear localization patterns that depend on factors required for generation of primary, but not secondary, siRNAs. In both wildtype and RNA interference (RNAi) mutants, viral dsRNA is observed in the cytoplasm associated with oRdRP, suggesting cytoplasmic virus replication hubs. Additionally, using antibodies to oRdRP, we observed spherical structures of ~1μm in diameter defined by oRdRP at their surface; over 75% of infected wildtype animals show these structures, which associate with mitochondria and autophagosomes in an antiviral RNAi- and autophagy-dependent manner, respectively. Our study defines new features of OV replication intermediates in wildtype animals, setting the stage for understanding their connection to the viral life cycle and host antiviral pathways.

Journal Article↗
PubMed2025

The mRNA export pathway licenses viral mimicry response and antitumor immunity by actively exporting nuclear retroelement transcripts.

Nuclear retroelement transcripts (RTs), which can be elicited both transcriptionally and posttranscriptionally, form double-stranded RNA (dsRNA) in cytosol to trigger the viral mimicry response (VMR) and antitumor immunity. However, the strength of the induced VMR varies tremendously across tumor types, and the underlying mechanisms remain poorly understood. Here, we demonstrate that the mRNA export pathway modulates the VMR through actively exporting nuclear RTs for cytosolic dsRNA formation after their induction. Tumor cells hijack this process for immune evasion through aberrant coactivator-associated arginine methyltransferase 1 (CARM1) expression. Mechanistically, we show that the cytoplasmic transportation of RTs by the mRNA export pathway is counteracted by the RNA exosome, which cleaves multiple transcripts within this pathway, including those encoding the essential DExD-box helicase 39A (DDX39A) and the adaptor protein ALYREF. CARM1 enhances the RNA exosome activity to attenuate the nuclear export of RTs by the mRNA export pathway through two synergistic mechanisms: (i) transcriptionally activating several RNA exosome components and (ii) posttranslationally methylating arginine 6 of the RNA exosome subunit EXOSC1, which protects it from proteasome-mediated degradation. Collectively, our study highlights the critical active regulatory role of the mRNA export pathway in transporting nuclear RTs into the cytosol for triggering the VMR and tumor immunity. Furthermore, we propose that enhancing the mRNA export pathway activity, either through CARM1 inhibition or RNA exosome modulation, could reinforce the therapeutic agent-induced VMR, thus holding the promise for overcoming tumor immune evasion and immunotherapy resistance.

Humans↗
PubMed1976

Regulated transcription of the genomes of defective virions and temperature-sensitive mutants of reovirus.

Defective reovirus, which lacks the largest (L1) of the 10 double-stranded (ds) RNA genomic segments, attaches to L cells and is uncoated in the same way as reovirus. The defective genome does not replicate in the cells, but it is transcribed. During the first 5 h after infection, three of the genomic segments, M3, S3, and S4, are more frequently transcribed than the remaining six segments. During the succeeding 5 h, there is a transition to a situation in which all nine segments are transcribed at the same relative frequencies. Since the class C ts mutation has been allocated to the L1 segment (Spandidos and Graham, 1975) the transcription of the C mutant genome was investigated in cells infected with it at the nonpermissive temperature, at which the parental genome does not replicate. Genomic segments L1, M3, S3, and S4 are predominantly transcribed at early times, and later all 10 segments are transcribed with the same relative frequencies. Transcription of the defective viral genome and the C mutant genome is therefore regulated in the same way as previously found for wild-type virus (Nonoyama, Millward, and Graham, 1974), and the regulation is independent of genome replication. Apparently the L1 segment function is involved in dsRNA synthesis but not in regulating the early to late transcription. It is suggested that a cellular repressor may be involved in this regulation and that derepression might be effected by one of the early viral gene products. Virion transcriptase activity was studied in vitro with cores prepared by chymotrypsin digestion of purified defective and standard virions. For both genomes the relative frequencies of transcription of the dsRNA segments are inversely proportional to their molecular weights. These results can be accounted for in a model that postulates each segment to be transcribed independently of the other. The same model with certain restrictions can describe the in vivo transcription of the viral genome.

Cell-Free System↗
PubMed2025

Argonaute 2 targets viral transcripts but not genomes of RNA viruses during antiviral RNA interference in Drosophila.

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we show that AGO2 is associated with ribosomes in control and virus infected cells. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes.

Animals↗
PubMed2026

IRES-like element-mediated translation of vsp1S4(-) suppresses BmCPV replication via RNAi antagonism.

Double-stranded RNA (dsRNA) viruses are thought to express proteins exclusively from their sense strand, while the antisense strand serves primarily as a replication template. Whether the antisense strand harbors hidden coding potential remains largely unexplored. Here, by integrating ribosome profiling and mass spectrometry, we identify a conserved 78-amino acid microprotein, vsp1S4(-), encoded by an antisense small open reading frame (sORFs) of the Bombyx mori cypovirus (BmCPV) genome. We demonstrate that vsp1S4(-) translation is driven by a previously unrecognized IRES-like element. Functional characterizations reveal that vsp1S4(-) localizes to the plasma membrane and acts as a negative regulator of viral replication. Mechanistically, vsp1S4(-) interacts directly with the viral RNAi suppressor NSP8, competitively disrupting the NSP8-AGO2 complex. This action restores the host's antiviral RNAi response, thereby limiting viral proliferation. Our findings challenge the conventional view of dsRNA virus coding capacity, unveil a novel viral immune evasion and replication control mechanism, and highlight antisense-encoded microproteins as potential targets for antiviral therapy.

Animals↗
PubMed2026

Genome characterization of two novel mitoviruses and a negative-sense single-stranded RNA mycovirus from the phytopathogenic fungus Clarireedia jacksonii.

Clarireedia jacksonii is a phytopathogenic fungus responsible for dollar spot disease in turfgrass worldwide. In this study, we characterized the complete genome sequences of three novel mycoviruses isolated from C. jacksonii isolate MBCT-836 using next-generation sequencing and the fragmented and primer-ligated dsRNA sequencing (FLDS) method. Two of these viruses, designated Clarireedia jacksonii mitovirus 1 (CjMV1) and Clarireedia jacksonii mitovirus 2 (CjMV2), possess positive-sense single-stranded RNA genomes of 2,575 bp and 2,856 bp, respectively. Both viruses contain a single open reading frame that utilizes the mitochondrial genetic code and encodes an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis placed CjMV1 and CjMV2 within the genera Unuamitovirus and Duamitovirus, respectively, in the family Mitoviridae. The third virus, Clarireedia jacksonii negative-stranded RNA virus 1 (CjNSV1), features a bisegmented negative-sense RNA genome consisting of a large segment (7,961 nt) encoding an RdRp with a conserved Bunya_RdRp domain, and a small segment (1,444 nt) encoding a protein showing homology to bunyavirus nucleocapsid proteins. Phylogenetic analysis revealed that CjNSV1 clusters with members of the proposed family Sclerobunyaviridae within the order Bunyavirales. To our knowledge, this study provides the first report of complete genome sequences of mycoviruses infecting C. jacksonii, expanding our understanding of the mycovirosphere in economically significant turfgrass pathogens.

Genome, Viral↗
PubMed2026

Exploring the potential of RNA interference (RNAi) in mosquito control: from mechanisms to molecular insights.

Mosquito-borne diseases represent a growing global health crisis, exacerbated by climate change and insecticide resistance. RNA interference (RNAi), a natural mechanism of gene silencing, offers a promising, target-specific alternative for mosquito control. This review explores the potential of RNAi to disrupt critical physiological processes, such as reproduction and disease transmission, thereby reducing vector populations and competence. We examine the mechanisms of RNAi, its application in combatting insecticide resistance, and recent advancements in delivery systems, including nanobody- and chitosan-based nanoparticles, which enhance the stability and uptake of double-stranded RNA (dsRNA) molecules. However, significant challenges remain, such as optimizing field-effective delivery methods and assessing potential off-target effects on non-target organisms. Continued innovation in RNAi technology is pivotal for developing sustainable and environmentally sound vector control strategies. This review synthesizes current research, highlighting the molecular insights, practical applications, and future directions for integrating RNAi into modern public health initiatives.

RNA Interference↗
PubMed2026

Evolutionary conservation of heat shock proteins in Blattodea and their roles in wing morphogenesis and ovarian development of Blattella germanica.

Heat shock proteins (Hsps) are essential molecular chaperones for protein homeostasis and stress responses. However, the Hsp repertoires and functions in Blattodea remain underexplored. Our genome-scale survey of nine Blattodea species revealed 37-46 conserved Hsp90, Hsp70, and DNAJ (Hsp40) genes, with DNAJ the most abundant and Hsp90 the least. Phylogenetic analysis confirmed the evolutionary conservation of three Hsp90, seven Hsp70, and 29 DNAJ subclades in Blattodea. Selection pressure analysis revealed predominant purifying selection (dN/dS ≪ 1) across lineages, strongest in DNAJ and highest in Hsp90 conservation. In Blattella germanica, expression of six representative BgHsp genes progressively increased during development, peaking in fifth-instar nymphs. Tissue expression profiling revealed that BgHspA1-2/3/4 were predominantly expressed in legs, BgDNAJB5 and BgHsp90AB1-2 were enriched in the fat body, and BgHsp90AB1 was highly expressed in the head. dsRNA injection targeting conserved Hsp gene regions achieved 61.9-94.1% knockdown of all six target genes. RNAi knockdown of six BgHsp genes disrupted wing morphogenesis, causing distinct phenotypes: wing whitening (56.7%, dsBgHspA1-4), unequal length (66.7%, dsBgHspA1-3; 76.7%, dsBgDNAJB5), and wing wrinkling (70%, dsBgHspA1-2; 63.3%, dsBgHsp90AB1; 76.7%, dsBgHsp90AB1-2). During ovarian formation, the developmental delay was most severe in the dsBgHsp90AB1 group, moderate in the dsBgHsp90AB1-2 and dsBgHspA1-2/3/4 groups, and weakest in the dsBgDNAJB5 group. Besides, knockdown significantly downregulated key developmental genes (apterous-a, nubbin, scalloped, ultrabithorax, wingless, and vitellogenin). These findings provide a reference for understanding the evolutionary patterns of Hsps in Blattodea, and offer mechanistic insights into the developmental regulation mediated by Hsps in this important public-health pest.

Animals↗
PubMed2024

A RNA Dodecahedral Cage Inside a Human Virus Plays a Dual Biological Role in Virion Assembly and Genome Release Control.

Human rhinoviruses (RV) are among the most frequent human pathogens. As major causative agents of common colds they originate serious socioeconomic problems and huge expenditure every year, and they also exacerbate severe respiratory diseases. No anti-rhinoviral drugs or vaccines are available so far. Antiviral drug design may benefit from an understanding of the role during the infectious cycle of the interactions in the virion between the capsid and the viral nucleic acid. The genomic RNA inside the human RV virion forms a dodecahedral cage made of 30 double-stranded RNA elements that interact with equivalent sites at the capsid inner wall. RNA dodecahedral cages also occur in distantly related insect and plant viruses. However, the functional role(s) of the interactions between any dodecahedral cage and the capsid remained to be established. Here we describe an extensive structure-function mutational analysis of the capsid-RNA dodecahedral cage interface in the RV virion, to dissect the role of the interactions between the capsid and the cage-forming RNA duplexes in: (i) infection by RV; (ii) virus biological fitness; (iii) virion assembly; (iv) virion stability; and (v) viral RNA uncoating. The results reveal that the capsid-bound dsRNA dodecahedral cage in the human RV virion is a multifunctional structural element. Two structurally overlapping subsets of RNA duplex-capsid interactions promote virus infectivity and biological fitness by respectively facilitating virion assembly or restraining the untimely, unproductive uncoating of the viral RNA genome. These results provide new insights into virion morphogenesis and genome uncoating, and have implications for antiviral drug design.

RNA, Viral↗
PubMed1976

The genetic control of DS-RNA virus-like particles associated with Saccharomyces cerevisiae killer yeast.

Previous results have indicated that laboratory strains of yeast possessing two types of dsRNA virus-like particles, V1 and V2, are capable of producing a killer toxin which kills sensitive strains. This paper reports on genetic crosses which correlate the presence of these particles both with a host nuclear gene +/-/mak1 and with the production of a killer toxin. The results show that: (i) V1 particles are inherited independently of the +/-/mak1 gene. (ii) V2 particles are present only in combination with those of V1 and the +/- allele of the +/-/mak1 nuclear gene.

Alleles↗
PubMed1977

Virus-like particles in Ustilago maydis: mutants with partial genomes.

Mutants with partial genomes for the virus-like particles of U. maydis were recovered following treatment with nitrosoguanidine. Examination of the properties retained by progeny of genetic crosses indicates that the 2.9 X 10(6) dalton component of double-stranded RNA contains the information for capsid formation and dsRNA replication. Other components appear to contain the information for killer function and immunity to killer. The use of such mutants for studies on the evolution of viruses with segmented genomes is discussed.

Basidiomycota↗