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PubMed1985

Isolation and characterization of mutants affecting functional domains of ColE1 RNAI.

The control of DNA replication initiation in the plasmid ColE1 is mediated by RNAI, a 108 nucleotide plasmid-encoded RNA that is entirely complementary to the 5'-terminal region of the replication primer RNA. RNAI acts in trans to inhibit primer maturation. Previously, we constructed a plasmid in which the ColE1 RNAI was separated from the primer and placed under transcriptional control of the Serratia marcesens tryptophan promoter. This plasmid provides RNAI in trans in vivo and mediates ColE1-type incompatibility. To determine the critical structural and functional domains of RNAI, we have undertaken a mutational analysis of the RNAI gene carried by this plasmid. We have selected mutants that no longer mediate ColE1-type incompatibility in trans. From the DNA sequences of 18 mutants we have identified mutations at nine new sites in RNAI. In addition, we have determined the secondary structural features of several mutant RNAI species and compared them to wild-type RNAI. Analysis of these mutations has revealed several key features of RNAI secondary structure and function. The domains of RNAI identified in this work which are essential for its function are: the single-stranded loop regions; the integrity of the double-stranded stems; and the single-stranded 5' terminus.

Colicins↗

PubMed2026

RNAi in the Rhizarian Phytopathogen Plasmodiophora brassicae: The Causal Agent of Clubroot Disease in Cruciferous Crops.

Although RNA interference (RNAi) is widespread and functionally important across eukaryotes, RNAi pathways are diverse or even lost in some lineages. Rhizaria represents a major and distinct eukaryotic supergroup that includes Plasmodiophora brassicae (Pb), the causal agent of cruciferous clubroot disease, yet RNAi in this lineage remains poorly understood. Here, we characterized an unusual RNAi pathway in Pb. Small RNA sequencing across five representative Pb life stages revealed abundant siRNAs and miRNAs characterized by a predominant 21-nt length, phased genomic distribution, 2-nt 3' overhangs, and a strong 5'-cytidine bias. Three Pb miRNAs were further validated by northern blotting and stem-loop RT-qPCR. Genome analysis identified two canonical AGO homologs, PbAGO1 and PbAGO2, but no Dicer homologs, except for an RNase III-containing Drosha-like protein, PbDRL. Functional analyses showed that PbAGO1 and PbAGO2 mediate gene silencing, whereas PbDRL is required for sRNA biogenesis. Further, the cell wall component chitin was identified from Pb zoosporangia during the early infection and RNAi interfering with its biosynthesis in transgenic plants of Arabidopsis and Brassica napus blocked Pb early infection and conferred broad-spectrum resistance. Our study uncovers an unusual RNAi pathway in Rhizaria and provides a promising strategy to control cruciferous clubroot disease.

Plasmodiophora brassicae↗

PubMed1986

Cloning and characterization of rat 4.5S RNAI genes.

Genomic clones containing genes for 4.5S RNAI, which is an abundant small nuclear RNA found in rodent cells, were obtained from a rat genomic library. Thirty-four clones that formed RNase A resistant hybrids with 3'-end-labeled 4.5S RNAI were isolated, and seven of them (clones lambda I39, lambda I41, lambda I42, lambda I51, lambda I106, lambda I123 and lambda I154) were characterized by sequencing and in vitro transcription. Clones lambda I41 and lambda I123 carry one and two genes, respectively, with identical sequences to that of 4.5S RNAI and are actively transcribed in vitro. However, the other five clones contain sequences that seem to be pseudogenes for 4.5S RNAI, since they have nucleotide substitutions or deletions in the sequence corresponding to 4.5S RNAI or are not transcribed. Four clones (lambda I39, lambda I42, lambda I106 and lambda I154) were found to have 13-18 nucleotide-long direct repeats flanking the 4.5S RNAI sequences. The genomic organization of the genes and their related sequences is discussed.

Animals↗

PubMed1988

Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII.

We have studied the interactions between the three molecules Rop, RNAI and RNAII that are involved in the regulatory mechanism controlling the replication of ColE1 plasmids. We show that it is possible to purify the two RNA molecules by passing an RNA mixture through an affinity column containing Rop immobilized to a solid support. The dissociation constants of the Rop-RNAI and Rop-RNAII complexes are of the order of 10(-4) M, several orders of magnitude higher than dissociation constants of stable protein-nucleic acid complexes (10(-10) M in the lambda repressor system). Although complete RNAI molecules have higher affinity, stem-and-loop I alone can also bind Rop, suggesting that this structure plays an important role in the interaction. Rop protects the stems of RNAI and RNAII from digestion by RNases while the sensitivity of the loops to digestion by RNase T1 is not affected by high concentrations of Rop. We propose a model for Rop-RNAI/RNAII interaction in which the dimeric protein acts as an adaptor between stem structures to position the two RNAs in the correct position for loop interaction.

Bacteriocin Plasmids↗

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↗

PubMed1991

The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo.

We show that the rate of degradation of RNAI, an anti-sense repressor of the replication primer RNAII, is a key element of control in the replication of ColE1-type plasmids in vivo. Cleavage of RNAI by RNAase E, a ribosomal RNA-processing enzyme encoded or controlled by the rne (also known as ams) locus, relieves repression by endonucleolytically converting RNAI to a very rapidly decaying product, pRNAI-5. A 5' triphosphate-terminated homolog of pRNAI-5 is degraded slowly and consequently inhibits replication. Nucleotide substitutions within the RNAase E cleavage sequence alter RNAI half-life and plasmid copy number, changing also the incompatibility phenotype. RNAI variants lacking the sequence cleaved by RNAase E are eliminated by growth rate-dependent degradation, resulting in growth-responsive control of plasmid replication and copy number.

Bacteriocin Plasmids↗

PubMed1991

Maintenance of pBR322-derived plasmids without functional RNAI.

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.

Bacteriocin Plasmids↗

PubMed1978

The effect of an immune RNA (RNAi) against Trypanosoma cruzi infection in mice.

Immune ribonucleic acid (RNAi) was extracted with phenol from the spleen of mice immunized with the avirulent PF strain of Trypanosoma cruzi. These preparations were able to induce immunocompetent cells to answer as a secondary response to later challenges with the virulent Y strain of the same parasite. The preparations of RNAi were"immunogenic" and free of proteins. The RNAi preparations were sensitive to pancreatic RNAase and lost their immune effect when pretreated with this enzyme. The injections of normal RNA (RNAn) obtained by the same method showed that this polymer acts as an immuno supressor or competitive agent. The electrophoretic profiles of the RNAi preparations in polyacrylamide gels showed normal and characteristic migration patterns (28S, 18S and 4.5S). These results demonstrate the development of an immune state against T. cruzi infection in mice injected with RNAi, in the absence of living parasites.

Animals↗

PubMed2026

Screening, optimization and artificial recombination of dsRNA fragments for RNAi-mediated pest resistance in Apolygus lucorum.

RNA interference (RNAi) is an eco-friendly strategy for pest management, with double-stranded RNA (dsRNA) as the core functional component. In this study, three RNAi target genes (Ubx, wupA and Dpp) with strong lethal effects on Apolygus lucorum were screened via microinjection. The 7-day cumulative mortalities were 56.67 ± 3.33% for dsUbx, 94.44 ± 1.11% for dswupA and 92.22 ± 1.11% for dsDpp. We optimized dsRNA sequences by removing conserved sequences in non-target organisms based on homology alignment and off-target risk analysis. The optimized fragments dswupA-OTE and dsDpp-OTE still exhibited high insecticidal activity, with 7-day cumulative mortalities of 77.78 ± 2.94% and 70.00 ± 1.93%, respectively. We also evaluated the effects of dsRNA length and target sites on RNAi efficiency and screened potent short dsRNA fragments. Novel artificially recombinant dsRNAs were constructed by assembling effective short fragments from different genes, which retained strong insecticidal activity despite shorter sequence length. This study verifies the feasibility of multi-target recombinant dsRNA for pest control and provides a theoretical basis for developing multi-gene RNAi technologies against A. lucorum.

Apolygus lucorum↗

PubMed1985

Adenovirus VA RNAI mediates a translational stimulation which is not restricted to the viral mRNAs.

The effect of adenovirus VA RNAI on the translation of mRNAs expressing the bacterial chloramphenicol acetyltransferase (CAT) enzyme was studied by a transient expression assay in 293 cells. The CAT activity was determined in extracts prepared from cells transfected with mixtures of plasmids encoding CAT and VA RNA. The results showed that VA RNAI co-transfection resulted in a significant increase in CAT expression from a variety of constructs. Thus, expression of CAT from a SV40 mRNA, a beta-globin mRNA and a chimeric mRNA containing the adenovirus-2 tripartite leader were all stimulated approximately 6-fold by VA RNAI. Based on these results we conclude that the tripartite leader sequence is not required for the VA RNA-mediated stimulation of translation. Our results indicate instead that VA RNAI probably functions as a general enhancer of mRNA translation. A2- to 3-fold stimulation of CAT expression was also obtained following transient expression of HeLa and CV-1 cells. The reduced efficiency was correlated with a 10- to 20-fold lower level of VA RNA expression in HeLa compared with 293 cells. Thus, it is likely that a product from region E1 indirectly enhances the translational efficiency by stimulating VA RNA transcription.

Acetyltransferases↗

PubMed1988

Effects of mutations in stem and loop regions on the structure and function of adenovirus VA RNAI.

Adenovirus virus-associated (VA) RNAI is required for efficient protein synthesis at late times of adenoviral infection, and in some other situations where double-stranded RNA (dsRNA) is present. It prevents inhibition of protein synthesis by a dsRNA-activated protein kinase and the secondary structure of VA RNAI is though to be important for its activity. To test this idea and to define structures and sequences responsible for VA RNAI activity, we constructed several mutant VA RNA genes and tested them in a transient expression assay. Activity is unaffected by deletions within a small region near the center of the gene, nt 72-85, but it is greatly diminished by deletion or substitution of sequences on the 3' side of this region. The structures of wild-type and mutant RNAs were examined by nuclease-sensitivity analysis. We propose a model for wild-type VA RNAI which differs from that predicted to be the most stable structure. Surprisingly disruption of the longest duplex region in the molecule is tolerated, provided that adjacent structural elements are not rearranged. However, perturbations of elements located in the center of the structure correlate well with loss of function.

Adenoviruses, Human↗

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↗

PubMed1986

A mechanism for the control of protein synthesis by adenovirus VA RNAI.

In the absence of VA RNAI, protein synthesis in adenovirus-infected HeLa cells fails because of defective initiation. Earlier work showed that the defect results from phosphorylation of the initiation factor elF-2 on its alpha subunit. We have identified the protein kinase responsible as the dsRNA-activated inhibitor of protein synthesis (DAI). DAI is present in uninfected state. It is activated in cells infected with the adenovirus mutant Ad5 dl331, which produces no VA RNAI, but not in cells infected with wild-type virus. Activation occurs during the late phase of infection with the mutant virus, and the activator appears to be dsRNA produced by symmetrical transcription of the viral genome. VA RNAI antagonizes the activation of DAI by dsRNA, but it cannot inhibit the activity of DAI once activated. We propose a mechanism for VA RNAI action based on its partially double-stranded nature.

Adenoviruses, Human↗

PubMed1986

Defective RNA splicing in the absence of adenovirus-associated RNAI.

We have analyzed late gene expression in 293 cells infected with an adenovirus type 5 mutant dl331, which is defective in production of the low molecular weight virus-associated (VA) RNAI. The results show that several steps in late gene expression are affected. In addition to the previously characterized defect in late mRNA translation, mutant infected cells also show an aberrant selection of RNA splice sites and a substantially reduced L2, L3, and L5 mRNA accumulation. Normal or even slightly elevated amounts of mRNA from region L1 are produced. However, the L1 pre-mRNA is spliced only to generate the mRNA encoding the Mr 52,000-55,000 polypeptide and no detectable mRNA for polypeptide IIIa. Cotransfection of a plasmid encoding VA RNAI complemented the splicing defect in trans, suggesting that the abnormalities are due to the absence of VA RNAI, rather than to a cis-acting change in the nuclear precursor RNA. In a HeLa cell variant, which allows late protein synthesis also in the absence of VA RNAI, because of a lack of eukaryotic initiation factor 2 alpha kinase expression, a normal repertoire of late mRNA was produced. We conclude that a soluble factor, most likely a late viral protein, controls differential RNA splicing and late mRNA accumulation during an adenovirus infection.

Adenoviruses, Human↗

PubMed1986

Characterization of a low-molecular-weight virus-associated (VA) RNA encoded by simian adenovirus type 7 which functionally can substitute for adenovirus type 5 VA RNAI.

Human adenoviruses (Ads), like Ad type 2 (Ad2) and Ad5, encode a low-molecular-weight RNA (designated virus-associated [VA] RNAI) which is required for the efficient translation of viral mRNAs late after infection. We cloned and characterized a VA RNA gene from simian adenovirus type 7 (SA7) which appears to have biological activity analogous to that of Ad2 VA RNAI. Thus, SA7 VA RNA stimulates protein synthesis in a transient expression assay and can also functionally substitute for VA RNAI during lytic growth of human Ad5. The SA7 genome encodes only one VA RNA species, in contrast to human Ad2, which encodes two distinct species. This RNA is transcribed by RNA polymerase III in the rightward direction from a gene located at about coordinate 30 on the viral genome, like its Ad2 counterparts. SA7 VA RNA shows only a limited primary sequence homology with the Ad2 VA RNAs (approximately 55%); the flanking sequences, in fact, are better conserved than the VA RNA gene itself. The predicted secondary structure of SA7 VA RNA is, however, very similar to that of Ad2 VA RNAI, inferring that the double-stranded nature rather than the primary sequence of VA RNA is important for its biological activity.

Adenoviridae↗

PubMed2026

Chitosan-dsRNA improves tissue stability and delivery for RNAi-mediated Varroa destructor control.

BACKGROUND: Varroa destructor is an ectoparasitic mite and a major threat to honey-bee colony health worldwide. RNA interference (RNAi) offers a potentially species-specific approach for mite control, but practical application is limited by double-stranded RNA (dsRNA) degradation and inefficient delivery to mites. This study evaluated coatomer protein I (COPI) complex subunits as RNAi targets and tested whether chitosan-based dsRNA formulation could improve dsRNA stability, tissue uptake, and delivery from honey-bees to mites. RESULTS: Direct microinjection of dsRNAs targeting COPB, COPD, and COPE significantly reduced target-gene expression and mite survival compared with the double-stranded green fluorescent protein (dsGFP) control, with 72-h survival rates of 8.0%, 12.7%, and 5.3%, respectively, compared with 40.7% in the control group (all log-rank P < 0.0001). Chitosan-conjugated dsRNA remained detectable for longer periods than naked dsRNA in honey-bee tissue fluids, and CNP-Cy3-dsGFP was detected in the honey-bee midgut and fat body. A qualitative fluorescence observation in V. destructor was consistent with host-to-mite dsRNA transfer. Ingestion of COP-targeted chitosan-dsRNAs reduced mite survival, whereas honey-bee survival and expression of honey-bee COP orthologs were not affected. In silico analysis detected no contiguous ≥19-nt matches between Varroa COP dsRNAs and the honey-bee transcriptome or genome. CONCLUSION: COPI subunits are promising RNAi targets in V. destructor, and chitosan formulation may improve dsRNA persistence and uptake while supporting honey-bee-mediated delivery to mites. These laboratory findings support further evaluation of chitosan-formulated dsRNA as a potentially species-selective strategy for Varroa management, while broader safety assessment and field validation remain necessary. © 2026 Society of Chemical Industry.

COPI complex↗

PubMed2025

Generation of Cdc20 RNAi-Sensitive Cell Lines to Study Mitotic Exit.

Accurate mitotic progression ensures the fidelity of genome passage. Cdc20 is a key mitotic regulator. It promotes mitotic exit by activating the anaphase-promoting complex or cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activating the spindle assembly checkpoint (SAC). Precise characterization of Cdc20 requires efficient depletion of endogenous Cdc20, which is extremely difficult to achieve by RNA interference (RNAi). This chapter describes the methodology to generate Cdc20 RNAi-sensitive cell lines with the help of CRISPR/Cas9 technology. These cell lines are highly sensitive to Cdc20 RNAi and provide a very useful tool for Cdc20 functionality investigation without the interference of endogenous Cdc20 protein. Similar strategy could be applied to other genes.

Cdc20 Proteins↗

PubMed2026

De novo transcriptome meta-analysis reveals candidate genes involved in life-stage transitions for RNAi-mediated management of the citrus root weevil (Diaprepes abbreviatus).

BACKGROUND: The citrus root weevil, Diaprepes abbreviatus, is a destructive agricultural pest for which molecular control options remain limited due to historically sparse genomic resources. Leveraging a comprehensive de novo transcriptome, we investigated developmental gene regulation across larval, pupal, and adult stages and identified essential targets for RNA interference (RNAi)-based intervention. RESULTS: Stage-resolved transcriptomic analyses revealed extensive transcriptional reprogramming associated with metabolism, detoxification, cuticle biosynthesis, endocrine signaling, and sensory perception. Among these, chitin synthase (DaCHS) emerged as a critical developmental gene, exhibiting pronounced up-regulation during late larval and pupal stages corresponding to intensive cuticle synthesis. Phylogenetic and structural analyses demonstrated that DaCHS is highly conserved among insects and retains canonical catalytic domains and transmembrane topology. Alpha Fold-based structural modeling and molecular docking confirmed stable interaction of DaCHS with its substrate, N-acetylglucosamine, supporting functional conservation of enzymatic activity. Oral delivery of DaCHS double-stranded RNA induced robust transcript suppression, leading to significant mortality and severe developmental defects, including larval and pupal abnormalities, and adults with disrupted wing and abdominal morphogenesis. CONCLUSION: These findings establish DaCHS as an indispensable gene for D. abbreviates development and validate transcriptome-guided RNAi as a powerful framework for target discovery. This work provides a strong molecular foundation for developing RNAi-based strategies that can be integrated into sustainable management programs for citrus root weevil control. © 2026 Society of Chemical Industry.

Animals↗