Search Results (129)

The genomes of viruses in the Allexivirus genus encode the p42 protein, which is considered the hallmark of the genus. The functions of p42 have not yet been studied experimentally and cannot be predicted based on sequence similarity, as p42-related proteins are not found among known cell or viral proteins. Here, p42 of Shallot virus X (ShVX), the type allexivirus, is demonstrated to be translated via a leaky scanning mechanism on a template comprising three “triple gene block” (TGB) transport genes and the p42 gene. Sequence analysis shows that this p42 expression mechanism is conserved in the vast majority of allexiviruses. p42 binds single-stranded RNA (ssRNA) but not double-stranded RNA (dsRNA) in vitro and localizes to the cytoplasm in association with microtubules and microtubule-bound bodies. In transient expression assays, p42 exhibits weak but detectable suppression of silencing induced by ssRNA but not by dsRNA. In addition, p42 suppresses silencing in the context of virus infection. Furthermore, p42 inhibits nonsense-mediated RNA decay (NMD) induced by a long 3′-terminal untranslated region of mRNA. Taken together, these findings provide initial evidence that the ShVX TGB/p42 gene module functions as a single genomic unit in terms of protein expression, that p42 acts as a suppressor of NMD and silencing, and that it may have multiple roles, while the precise biological significance of p42 in these roles remains to be experimentally confirmed. Full article

Retroviruses are single-stranded RNA viruses that package two copies of their positively stranded RNA genomes as a non-covalent dimer into newly formed virions. This process is evolutionarily conserved, and disruption of genome dimerization results in production of non-infectious virus particles. Genome dimers can be packaged as homodimers, containing two identical RNAs, or heterodimers, consisting of two genetically distinct copies. Genome dimerization generates genetic diversity, and different retroviruses have preferences for the type of genome dimers packaged into virions. We developed a novel imaging approach to specifically label and detect retroviral genome heterodimers in cells using a modified bimolecular fluorescence complementation (BiFC) technique. This method utilizes viral genomes encoding two different RNA stem-loop cassettes that each specifically binds to an RNA-binding protein conjugated to a split fluorophore. When two genetically different genomes are within close proximity, the fluorophore halves come together to reconstitute fluorescence. These BiFC-labeled RNA dimers can be visualized and tracked in living cells and interact with retroviral Gag proteins. This method has the advantage of low background fluorescence and can be applied to the study of dimeric or double-stranded RNAs of viruses and other organisms. Full article

Tomato spotted wilt virus (TSWV) is a highly destructive plant pathogen and transmitted by several thrips including the western flower thrips, Frankliniella occidentalis. A structural N protein encoded in the viral genome represents the nucleocapsid protein by binding to the viral RNA genome. However, it remains unknown how the RNA-binding protein specifically interacts with the viral RNA from host RNAs in the target cells. To study the molecular basis of N function, we produced the protein in Escherichia coli and the resulting purified recombinant protein was used to investigate the protein–RNA interactions. The recombinant N protein migrated on agarose gel to the anode in the electric field due to its high basic isoelectric point. This electrostatic property led N protein to bind to DNA as well as RNA. It also bound to both single-stranded (ssRNA) and double-stranded RNA (dsRNA). However, when the total RNA was extracted from plant tissues collected from TSWV-infected host, the RNA extract using the recombinant N protein was much richer in the TSWV genome compared to that without the protein. To investigate the specificity of N protein to ssRNA, the three-dimensional structure was predicted using the AlphaFold program and showed its trimeric oligomerization with the binding pocket for ssRNA. This was supported by the differential susceptibility of N protein with ssRNA and dsRNA against RNase attack. Furthermore, a thermal shift assay to analyze the RNA and protein interaction showed that ssRNA strongly interacted with N protein compared to dsRNA. In addition, the N gene was expressed along with the multiplication of the viral RNA genome segments from the segment-specific fluorescence in situ hybridization analysis in different tissues during different developmental stages of the virus-infected F. occidentalis. These results suggest that the functional trimeric N proteins bind to the viral RNA to form a basic nucleocapsid structure at a specific virus-replicating compartment within the host cells. Full article

Background/Objectives: PARP inhibitors (PARPi) are pivotal to treating homologous recombination repair-deficient (HRD) cancers, particularly BRCA1/2-mutated ovarian and breast cancers. However, most ovarian and breast cancers harbor wild-type (WT) BRCA1/2, limiting PARPi eligibility. This study aims to identify an approved drug that could induce a BRCAness phenotype, thereby sensitizing WT BRCA cancers to PARPi. Methods: Ovarian and breast cancer cell lines with WT BRCA1/2 were treated with ivosidenib. HR repair efficiency was assessed via RAD51 foci formation and reporter assays. Synthetic lethality with PARPi was evaluated using viability and colony formation assays. Mechanistic studies included RNA-binding protein pulldown, co-immunoprecipitation, and functional analyses of DNA repair pathways. YTHDC2′s role in HR was investigated through siRNA knockdown and rescue experiments. Results: Ivosidenib significantly reduced HR repair efficiency and sensitized cells to PARPi, inducing synthetic lethality. Mechanistically, ivosidenib directly bound YTHDC2, an m6A reader critical for HR. This interaction disrupted YTHDC2′s ability to promote DNA double-strand break repair via HR, evidenced by impaired recruitment of repair proteins (e.g., BRCA1, RAD51) and accumulation of DNA damage (γH2AX foci). YTHDC2 knockdown phenocopied ivosidenib effects, while overexpression rescued HR defects. Conclusions: Ivosidenib induces BRCAness in WT BRCA ovarian and breast cancers by targeting YTHDC2, thereby suppressing HR repair and enhancing PARPi sensitivity. This uncovers a novel, metabolism-independent mechanism of ivosidenib, repositioning it as a therapeutic agent for HRD tumors. These findings propose a strategy to expand PARPi eligibility to WT BRCA cancers, addressing a critical unmet need in oncology. Full article

In plants, microRNAs (miRNAs) and their target genes have been demonstrated to form an essential component of the molecular response to salt stress. In Arabidopsis thaliana (Arabidopsis), DOUBLE-STRANDED RNA BINDING1 (DRB1) and DRB2 are required to produce specific miRNA populations throughout normal development and in response to abiotic stress. The phenotypic and physiological assessment of 15-day-old wild-type Arabidopsis seedlings, and of the drb1 and drb2 mutants following a 7-day period of salt stress, revealed the drb2 mutant to be more sensitive to salt stress than the drb1 mutant. However, the assessment of miRNA abundance and miRNA target gene expression showed that the ability of both drb mutants to mount an appropriate miRNA-mediated molecular response to salt stress is defective. Furthermore, molecular profiling also showed that DRB1 and DRB2 are both required for miRNA production during salt stress, and that both a target transcript cleavage mode and a translational repression mode of RNA silencing are required to appropriately regulate miRNA target gene expression as part of the molecular response of Arabidopsis to salt stress. Taken together, the phenotypic, physiological, and molecular analyses performed here clearly show that all components of the miRNA pathway must be fully functional for Arabidopsis to mount an appropriate miRNA-mediated molecular response to salt stress. Full article

The double-stranded RNA editing enzyme ADAR1 connects two forms of genetic programming, one based on codons and the other on flipons. ADAR1 recodes codons in pre-mRNA by deaminating adenosine to form inosine, which is translated as guanosine. ADAR1 also plays essential roles in the immune defense against viruses and cancers by recognizing left-handed Z-DNA and Z-RNA (collectively called ZNA). Here, we review various aspects of ADAR1 biology, starting with codons and progressing to flipons. ADAR1 has two major isoforms, with the p110 protein lacking the p150 Zα domain that binds ZNAs with high affinity. The p150 isoform is induced by interferon and targets ALU inverted repeats, a class of endogenous retroelement that promotes their transcription and retrotransposition by incorporating Z-flipons that encode ZNAs and G-flipons that form G-quadruplexes (GQ). Both p150 and p110 include the Zβ domain that is related to Zα but does not bind ZNAs. Here we report strong evidence that Zβ binds the GQ that are formed co-transcriptionally by ALU repeats and within R-loops. By binding GQ, ADAR1 suppresses ALU-mediated alternative splicing, generates most of the reported nonsynonymous edits and promotes R-loop resolution. The recognition of the various alternative nucleic acid conformations by ADAR1 connects genetic programming by flipons with the encoding of information by codons. The findings suggest that incorporating G-flipons into editmers might improve the therapeutic editing efficacy of ADAR1. Full article

Banana streak GF virus (BSGFV) is the extremely dangerous monopartite badnavirus (genus, Badnavirus; family, Caulimoviridae) of banana (Musa acuminata AAA Group) that imposes a serious threat to global banana production. The BSGFV causes a devastating pandemic in banana crops, transmitted by deadly insect pest mealybug vectors and replicated through an RNA intermediate. The BSGFV is a reverse-transcribing DNA virus that has a monopartite open circular double-stranded DNA (dsDNA) genome with a length of 7325 bp. RNA interference (RNAi) is a natural mechanism that has revolutionized the target gene regulation of various organisms to combat virus infection. The current study aims to locate the potential target binding sites of banana-encoded microRNAs (mac-miRNAs) on the BSGFV-dsDNA-encoded mRNAs based on three algorithms, RNA22, RNAhybrid and TAPIR. Mature banana (2n = 3x = 33) miRNAs (n = 32) were selected and hybridized to the BSGFV genome (MN296502). Among the 32 targeted mature locus-derived mac-miRNAs investigated, two banana mac-miRNA homologs (mac-miR162a and mac-miR172b) were identified as promising naturally occurring biomolecules to have binding affinity at nucleotide positions 5502 and 9 of the BSGFV genome. The in silico banana-genome-encoded mac-miRNA/mbg-miRNA-regulatory network was developed with the BSGFV—ORFs using Circos software (version 0.69-9) to identify potential therapeutic target proteins. Therefore, the current work provides useful biological material and opens a new range of opportunities for generating BSGFV-resistant banana plants through the genetic manipulation of the selected miRNAs. Full article

The tripartite-motif protein 56 (TRIM56) is a RING-type E3 ubiquitin ligase whose functions were recently beginning to be unveiled. While the physiological role(s) of TRIM56 remains unclear, emerging evidence suggests this protein participates in host innate defense mechanisms that guard against viral infections. Interestingly, TRIM56 has been shown to pose a barrier to viruses of distinct families by utilizing its different domains. Apart from exerting direct, restrictive effects on viral propagation, TRIM56 is implicated in regulating innate immune signaling pathways that orchestrate type I interferon response or autophagy, through which it indirectly impacts viral fitness. Remarkably, depending on viral infection settings, TRIM56 either operates in a canonical, E3 ligase-dependent fashion or adopts an enzymatically independent, non-canonical mechanism to bolster innate immune signaling. Moreover, the recent revelation that TRIM56 is an RNA-binding protein sheds new light on its antiviral mechanisms against RNA viruses. This review summarizes recent advances in the emerging roles of TRIM56 in innate antiviral immunity. We focus on its direct virus-restricting effects and its influence on innate immune signaling through two critical pathways: the endolysosome-initiated, double-stranded RNA-sensing TLR3-TRIF pathway and the cytosolic DNA-sensing, cGAS-STING pathway. We discuss the underpinning mechanisms of action and the questions that remain. Further studies understanding the complexity of TRIM56 involvement in innate immunity will add to critical knowledge that could be leveraged for developing antiviral therapeutics. Full article

DOUBLE-STRANDED RNA BINDING (DRB) proteins DRB1, DRB2, and DRB4 are essential for microRNA (miRNA) production in Arabidopsis thaliana (Arabidopsis) with miR160, and its target genes, AUXIN RESPONSE FACTOR10 (ARF10), ARF16, and ARF17, forming an auxin responsive miRNA expression module crucial for root development. Methods: Wild-type Arabidopsis plants (Columbia-0 (Col-0)) and the drb1, drb2, and drb12 mutants were treated with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), and the miR160-mediated response of these four Arabidopsis lines was phenotypically and molecularly characterized. Results: In 2,4-D-treated Col-0, drb1 and drb2 plants, altered miR160 abundance and ARF10, ARF16, and ARF17 gene expression were associated with altered root system development. However, miR160-directed molecular responses to treatment with 2,4-D was largely defective in the drb12 double mutant. In addition, via profiling of molecular components of the miR160 expression module in the roots of the drb4, drb14, and drb24 mutants, we uncovered a previously unknown role for DRB4 in regulating miR160 production. Conclusions: The miR160 expression module forms a central component of the molecular and phenotypic response of Arabidopsis plants to exogenous auxin treatment. Furthermore, DRB1, DRB2, and DRB4 are all required in Arabidopsis roots to control miR160 production, and subsequently, to appropriately regulate ARF10, ARF16, and ARF17 target gene expression. Full article

Arabidopsis thaliana (Arabidopsis) double-stranded RNA binding (DRB) proteins DRB1, DRB2 and DRB4 perform essential roles in microRNA (miRNA) production, with many of the produced miRNAs mediating aspects of the molecular response of Arabidopsis to abiotic stress. Exposure of the drb1, drb2 and drb4 mutants to mannitol stress showed drb2 to be the most sensitive to this form of osmotic stress. Profiling of the miRNA landscapes of mannitol-stressed drb1, drb2 and drb4 seedlings via small RNA sequencing, and comparison of these to the profile of mannitol-stressed wild-type Arabidopsis plants, revealed that the ability of the drb1 and drb2 mutants to mount an appropriate miRNA-mediated molecular response to mannitol stress was defective. RT-qPCR was next used to further characterize seven miRNA/target gene expression modules, with this analysis identifying DRB1 as the primary DRB protein required for miR160, miR164, miR167 and miR396 production. In addition, via its antagonism of DRB1 function, DRB2 was shown by RT-qPCR to play a secondary role in regulating the production of these four miRNAs. This analysis further showed that DRB1, DRB2 and DRB4 are all required to regulate the production of miR399 and miR408, and that DRB4 is the primary DRB protein required to produce the non-conserved miRNA, miR858. Finally, RT-qPCR was used to reveal that each of the seven characterized miRNA/target gene expression modules responded differently to mannitol-induced osmotic stress in each of the four assessed Arabidopsis lines. In summary, this research has identified mannitol-stress-responsive miRNA/target gene expression modules that can be molecularly manipulated in the future to generate novel Arabidopsis lines with increased tolerance to this form of osmotic stress. Full article

Cry2Ab is a significant alternative Bacillus thuringiensis (Bt) protein utilized for managing insect resistance to Cry1 toxins and broadening the insecticidal spectrum of crops containing two or more Bt genes. Unfortunately, the identified receptors fail to fully elucidate the mechanism of action underlying Cry2Ab. Previous studies have demonstrated the involvement of vacuolar H⁺-ATPase subunits A, B, and E (V-ATPase A, B, and E) in Bt insecticidal activities. The present study aims to investigate the contribution of V-ATPase C to the toxicities of Cry2Ab against Helicoverpa armigera. The feeding of Cry2Ab in H. armigera larvae resulted in a significant decrease in the expression of V-ATPase C. Further investigations confirmed the interaction between V-ATPase C and activated Cry2Ab protein according to Ligand blot and homologous and heterologous competition assays. Expressing endogenous HaV-ATPase C in Sf9 cells resulted in an increase in Cry2Ab cytotoxicity, while the knockdown of V-ATPase C by double-stranded RNAs (dsRNA) in midgut cells decreased Cry2Ab cytotoxicity. Importantly, a higher toxicity of the mixture containing Cry2Ab and V-ATPase C against insects was also observed. These findings demonstrate that V-ATPase C acts as a binding receptor for Cry2Ab and is involved in its toxicity to H. armigera. Furthermore, the synergy between V-ATPase C protein and Cry2Ab protoxins provides a potential strategy for enhancing Cry2Ab toxicity or managing insect resistance. Full article

The brown planthopper (Nilaparvata lugens) is a major pest threatening global rice production, significantly reducing yields annually. As N. lugens increasingly develops resistance to conventional control methods, such as chemical pesticides, there is an urgent need for innovative and sustainable pest management strategies. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a key protein involved in mRNA 3′ end processing, yet its function in N. lugens remains poorly understood. This study aims to elucidate the role of CPSF30 in the growth and development of N. lugens and evaluate its potential as a target for RNA interference (RNAi)-based pest control strategies. We cloned and characterized the cDNA sequence of NlCPSF30, which encodes a protein of 341 amino acids containing five CCCH zinc-finger domains and two CCHC zinc-knuckle domains. Sequence alignment revealed that NlCPSF30 is highly conserved among insect species, particularly in the zinc-finger domains essential for RNA binding and processing. Phylogenetic analysis showed that NlCPSF30 is closely related to CPSF30 proteins from other hemipteran species. Expression analysis indicated that NlCPSF30 is most highly expressed in the fat body and during the adult stage, with significantly higher expression in females than in males. RNAi-mediated silencing of NlCPSF30 in third-instar nymphs resulted in severe phenotypic abnormalities, including disrupted molting and increased mortality following injection of double-stranded RNA (dsRNA) targeting NlCPSF30. Moreover, it influenced the expression of genes associated with hormone regulation, namely NlHry, NlE93, and NlKr-h1. These results suggest that NlCPSF30 is integral to critical physiological processes, with its disruption leading to increased mortality. Our findings identify NlCPSF30 as an essential gene for N. lugens’ survival and a promising target for RNAi-based pest management strategies. This study provides a valuable molecular target and theoretical insights for developing RNAi-based control methods against N. lugens. Full article

Background: Transactivation Response Element RNA-binding Protein (TRBP2) is a double-stranded RNA-binding protein widely known for its critical contribution to RNA interference (RNAi), a conserved mechanism of gene-expression regulation mediated through small non-coding RNA moieties (ncRNAs). Nevertheless, TRBP2 has also proved to be involved in other molecular pathways and biological processes, such as cell growth, organism development, spermatogenesis, and stress response. Mutations or aberrant expression of TRBP2 have been previously associated with diverse human pathologies, including Alzheimer’s disease, cardiomyopathy, and cancer, with TRBP2 playing an essential role(s) in proliferation, invasion, and metastasis of tumor cells. Methods: Hence, the present study aims to investigate, via employment of advanced flow cytometry, immunofluorescence, cell transgenesis and bioinformatics technologies, new, still elusive, functions and properties of TRBP2, particularly regarding its cell cycle-specific control during cancer cell division. Results: We have identified a novel, mitosis-dependent regulation of TRBP2 protein expression, as clearly evidenced by the lack of its immunofluorescence-facilitated detection during mitotic phases, in several human cancer cell lines of different tissue origin. Notably, the obtained TRBP2-downregulation patterns seem to derive from molecular mechanisms that act independently of oncogenic activities (e.g., malignancy grade), metastatic capacities (e.g., low versus high), and mutational signatures (e.g., p53^−/− or p53^ΔΥ126) of cancer cells. Conclusions: Taken together, we herein propose that TRBP2 serves as a novel cell cycle-dependent regulator, likely exerting mitosis-suppression functions, and, thus, its mitosis-specific downregulation can hold strong promise to be exploited for the efficient and successful prognosis, diagnosis, and (radio-/chemo-)therapy of diverse human malignancies, in the clinic. Full article

Background: In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and quantitation methods, such as gel electrophoresis, ELISA, or homogeneous time-resolved fluorescence (HTRF), have low sensitivity or are time-consuming. A recently published lateral flow immunoassay (LFSA) was shown to be fast, but it lacks the sensitivity for dsRNA with uridine modifications. Methods: In this study, we provided a possible explanation for the reduced sensitivity of existing quantitation methods for dsRNA with modified uridines by characterizing the binding affinities of commonly used anti-dsRNA antibodies. Then, a rapid and sensitive biolayer interferometry (BLI) dsRNA detection assay utilizing Flock House Virus (FHV) B2 protein was developed to overcome the challenges in dsRNA detection and the reduced sensitivity. Results: This assay allows the detection of dsRNA with different uridine modifications (ψ, m1ψ, 5 moU) with similar sensitivity as dsRNA without modification. Furthermore, we demonstrated this method can be used to quantify both short and long dsRNA, as well as hairpin-structured dsRNA, providing a more comprehensive detection for dsRNA impurities. Moreover, we applied this assay to monitor dsRNA removal through a purification process. Conclusions: Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA. Full article

Inosine is a nucleotide resulting from the deamination of adenosine in RNA. This chemical modification process, known as RNA editing, is typically mediated by a family of double-stranded RNA binding proteins named Adenosine Deaminase Acting on dsRNA (ADAR). While the presence of ADAR orthologs has been traced throughout the evolution of metazoans, the existence and extension of RNA editing have been characterized in a more limited number of animals so far. Undoubtedly, ADAR-mediated RNA editing plays a vital role in physiology, organismal development and disease, making the understanding of the evolutionary conservation of this phenomenon pivotal to a deep characterization of relevant biological processes. However, the lack of direct high-throughput methods to reveal RNA modifications at single nucleotide resolution limited an extended investigation of RNA editing. Nowadays, these methods have been developed, and appropriate bioinformatic pipelines are required to fully exploit this data, which can complement existing approaches to detect ADAR editing. Here, we review the current literature on the “bioinformatics for inosine” subject and we discuss future research avenues in the field. Full article