Search Results (43)

In Drosophila gonads, transposable elements (TEs) are repressed by the Piwi-interacting RNA (piRNA) pathway operating both co-transcriptionally and post-transcriptionally. In the non-gonadal tissues, TEs are mainly repressed by the short interfering RNA (siRNA) pathway with Argonaute 2 (Ago2) functioning as an effector protein. It is generally assumed that this pathway acts at the post-transcriptional level. However, recent data point to its possible involvement in co-transcriptional silencing as well. Here, using DamID, we found a drastic decrease in HP1a on TEs (especially on the LTR-containing retrotransposons) and other heterochromatin regions in Ago2-mutant Drosophila brain. HP1a reduction is accompanied by the increased chromatin accessibility of TEs, indicating their derepression. Accordingly, several LTR-containing retrotransposons were up-regulated in the larval brain of Ago2 mutants. Moreover, upon the knock-down of lamin Dm0 in neurons, HP1a was increased predominantly on the same set of TEs that had reduced HP1a binding in Ago2 mutants. We hypothesize that, since Ago2 was localized to the common complex with lamin Dm0, the depletion of the latter may release Ago2 in the nucleoplasm, thus enhancing the recruitment of HP1a on TEs. Our findings support the hypothesis that TEs in the Drosophila brain are silenced, in part, through Ago2-mediated recruitment of HP1a. Full article

MicroRNAs (miRNAs) are key post-transcriptional regulators controlling gene expression across several cellular processes, including development, proliferation, and apoptosis. Their biogenesis involves a multi-step pathway, including the processing of primary transcripts and the assembly of the RNA-Induced Silencing Complex (RISC) with Argonaute (AGO) proteins at its core. This review provides a comprehensive overview of the molecular dynamics of miRNA-loaded RISC (miRISC), focusing on the post-translational modifications, the interactors of AGOs and the mechanisms that fine-tune and coordinate miRISC activity. The composition of miRISC influences AGO stability, localization, and silencing efficiency, thereby maintaining cellular homeostasis and development and mediating the response to various types of cellular stress. Uncommon regulatory mechanisms, including AGO modifications during, e.g., hypoxia or Type 2 T cell responses and miRISC functionality, with myriad RNA-binding proteins (RBPs), will be discussed. This review aims at highlighting the recent advances in the understanding of the intricate regulation of miRISC-driven gene silencing. Full article

Contact unmodified antisense DNA biotechnology (CUADb), developed in 2008, employs short antisense DNA oligonucleotides (oligos) as a novel approach to insect pest control. These oligonucleotide-based insecticides target pest mature rRNAs and/or pre-rRNAs and have demonstrated high insecticidal efficacy, particularly against sap-feeding insect pests, which are key vectors of plant DNA viruses and among the most economically damaging herbivorous insects. To further explore the potential of CUADb, this study evaluated the insecticidal efficacy of short 11-mer antisense DNA oligos against Coccus hesperidum, in comparison with long 56-mer single-stranded and double-stranded DNA sequences. The short oligos exhibited higher insecticidal activity. By day 9, the highest mortality rate (97.66 ± 4.04%) was recorded in the Coccus-11 group, while the most effective long sequence was the double-stranded DNA in the dsCoccus-56 group (77.09 ± 6.24%). This study also describes the architecture of the DNA containment (DNAc) mechanism, highlighting the intricate interactions between rRNAs and various types of DNA oligos. During DNAc, the Coccus-11 treatment induced enhanced ribosome biogenesis and ATP production through a metabolic shift from carbohydrates to lipid-based energy synthesis. However, this ultimately led to a ‘kinase disaster’ due to widespread kinase downregulation resulting from insufficient ATP levels. All DNA oligos with high or moderate complementarity to target rRNA initiated hypercompensation, but subsequent substantial rRNA degradation and insect mortality occurred only when the oligo sequence perfectly matched the rRNA. Both short and long oligonucleotide insecticide treatments led to a 3.75–4.25-fold decrease in rRNA levels following hypercompensation, which was likely mediated by a DNA-guided rRNase, such as RNase H1, while crucial enzymes of RNAi (DICER1, Argonaute 2, and DROSHA) were downregulated, indicating fundamental difference in molecular mechanisms of DNAc and RNAi. Consistently, significant upregulation of RNase H1 was detected in the Coccus-11 treatment group. In contrast, treatment with random DNA oligos resulted in only a 2–3-fold rRNA decrease, consistent with the normal rRNA half-life maintained by general ribonucleases. These findings reveal a fundamental new mechanism of rRNA regulation via complementary binding between exogenous unmodified antisense DNA and cellular rRNA. From a practical perspective, this minimalist approach, applying short antisense DNA dissolved in water, offers an effective, eco-friendly and innovative solution for managing sternorrhynchans and other insect pests. The results introduce a promising new concept in crop protection: DNA-programmable insect pest control. Full article

Myocardial injury is a critical determinant of prognosis in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, its underlying mechanisms remain incompletely understood. In this study, we examined the effects of SARS-CoV-2-derived RNA fragments on human cardiomyocytes. We identified a 19-nucleotide sequence within the viral genome that shares complete sequence homology with the human F1F0 ATP synthase subunit alpha gene (ATP5A). This sequence was found to associate with Argonaute 2 (AGO2) and downregulate ATP5A expression via a mechanism analogous to RNA interference. Consequently, oxidative phosphorylation was suppressed in cardiomyocytes, leading to impaired myocardial maturation and the emergence of heart failure-like phenotypes. Notably, exosome-mimetic liposomal delivery of this RNA fragment to cardiomyocytes reproduced the ATP5A-suppressive effect. These findings suggest that SARS-CoV-2-derived RNA fragments may contribute to myocardial injury through the siRNA-like modulation of mitochondrial gene expression. Further validation in animal models and patient-derived materials is warranted. Full article

Small RNAs (sRNAs) control a wide range of development and physiological pathways in plants. To address the response of sRNA biogenesis to drought stress, we identified sRNA biogenesis genes, including 11 encoding argonautes (AGO), 8 encoding Dicer-like proteins (DCL), and 9 encoding RNA-dependent RNA polymerases (RDR) in the peach genome. Notably, the largest numbers of sRNA biogenesis genes are located to chromosome 1. The PAZ, PIWI, and MID domains were identified in PpAGOs, while the ribonuclease IIIa and IIIb domains were characterized in PpDCLs. The RDRP domain was recognized in PpRDRs. Orthologous similarity and collinearity analyses between Arabidopsis and peach revealed 5, 1, and 2 collinear blocks in AGOs, DCLs, and RDRs, respectively. Moreover, 41, 40, and 42 cis-acting elements were located in the promoters of PpAGOs, PpDCLs, and PpRDRs, respectively, with the majority related to drought stress response. Analysis of RNA sequencing (RNA-seq) data revealed that sRNA biogenesis genes were involved in drought stress response in different tissues. Furthermore, the expression of candidate genes was verified in two peach cultivars, Beijing 2-7 (BJ2-7) and Sinai (SN), which are tested as drought-tolerant and sensitive cultivars, respectively, based on the physiological and biochemical analyses, which revealed that the Chinese peach cultivar ‘BJ2-7’ exhibits greater drought resistance compared to the Egyptian peach cultivar ‘SN’. Interestingly, the expression of PpAGO2b, PpDCL2b, PpDCL4, and PpRDR4 genes was induced in ‘BJ2-7’ but inhibited in ‘SN’ under drought stress. Overall, this study provides insight into the roles of sRNA biogenesis genes in response to drought stress in peach. Full article

During infection, adenoviruses inhibit the cellular RNA interference (RNAi) machinery by saturating the RNA-induced silencing complex (RISC) of the host cells with large amounts of virus-derived microRNAs (mivaRNAs) that bind to the key component of the complex, Argonaute 2 (AGO2). In the present study, we investigated AGO2 as a prominent player at the intersection between human adenovirus 5 (HAdV-5) and host cells because of its ability to interfere with the HAdV-5 life cycle. First, the ectopic expression of AGO2 had a detrimental effect on the ability of the virus to replicate. In addition, in silico and in vitro analyses suggested that endogenous microRNAs (miRNAs), particularly hsa-miR-7-5p, have similar effects. This miRNA was found to be able to target the HAdV-5 DNA polymerase mRNA. The inhibitory effect became more pronounced upon overexpression of AGO2, likely due to elevated AGO2 levels, which abolished the competition between cellular miRNAs and mivaRNAs for RISC incorporation. Collectively, our data suggest that endogenous miRNAs would be capable of significantly inhibiting viral replication if adenoviruses had not developed a mechanism to counteract this function. Eventually, AGO2 overexpression-mediated relief of the RISC-saturating action of mivaRNAs strongly enhanced the effectiveness of artificial miRNAs (amiRNAs) directed against the HAdV-5 preterminal protein (pTP) mRNA, suggesting a substantial benefit of co-expressing amiRNAs and AGO2 in RNAi-based strategies for the therapeutic inhibition of adenoviruses. Full article

Argonauts or paper nautiluses are pelagic octopod cephalopods with a cosmopolitan distribution in tropical and subtropical waters around the world. Unlike other species of octopus, these are characterized by the fact that the female has a shell that serves as the breeding chamber for the eggs. Over time, this structure has been used as a taxonomic diagnostic character, causing problems in the systematics of this genus, with around 50 synonymies reported. Only two species, Argonauta argo and A. nouryi, have been reported in the Northern Humboldt Current System; however, there is taxonomic uncertainty regarding these species, which is reflected in the paralarvae (the first stage of life after hatching). In the paralarvae, the chromatophore patterns are considered to be conservative and reliable taxonomic characteristics. The objective of this study is to demonstrate the extensive variability in the chromatophore arrangement of Argonauta paralarvae in the Northern Humboldt Current using DNA barcoding and five species delimitation models. Our results include up to 11 different paralarvae morphotypes according to the pattern of chromatophores (number and arrangement) and 2 shell morphotypes. Species delimitation methods divided the 13 Argonauta morphotypes into two consensus molecular taxonomic units (MOTUs), A. argo and A. nouryi. Additionally, the results revealed an extensive morphological variability in the paralarvae and female shells of A. nouryi, demonstrating the importance of molecular data in studies involving species with different life stages, especially when this extensive morphological variability obscures conventional analyses. Full article

Varroa destructor mites and the viruses it vectors are two major factors leading to high losses of honey bees (Apis mellifera) colonies worldwide. However, honey bees in some African countries show resilience to varroa infestation and/or virus infections, although little is known about the mechanisms underlying this resilience. In this study, we investigated the expression profiles of some key molecular markers involved in olfactory sensing and RNA interference, as these processes may contribute to the bees’ resilience to varroa infestation and virus infection, respectively. We found significantly higher gene expression of the odorant binding protein, OBP14, in the antennae of Ethiopian bees compared to Belgian bees. This result suggests the potential of OBP14 as a molecular marker of resilience to mite infestation. Scanning electron microscopy showed no significant differences in the antennal sensilla occurrence and distribution, suggesting that resilience arises from molecular processes rather than morphological adaptations. In addition, seven RNAi genes were upregulated in the Ethiopian honey bees and three of them—Dicer-Drosha, Argonaute 2, and TRBP2—were positively correlated with the viral load. We can conclude that the antiviral immune response was triggered when bees were experiencing severe viral infection and that this might contribute to the bees’ resilience to viruses. Full article

Hypoxia, or low oxygen tension, is frequently found in highly proliferative solid tumors such as anaplastic thyroid carcinoma (ATC) and is believed to promote resistance to chemotherapy and radiation. Identifying hypoxic cells for targeted therapy may thus be an effective approach to treating aggressive cancers. Here, we explore the potential of the well-known hypoxia-responsive microRNA (miRNA) miR-210-3p as a cellular and extracellular biological marker of hypoxia. We compare miRNA expression across several ATC and papillary thyroid cancer (PTC) cell lines. In the ATC cell line SW1736, miR-210-3p expression levels indicate hypoxia during exposure to low oxygen conditions (2% O₂). Furthermore, when released by SW1736 cells into the extracellular space, miR-210-3p is associated with RNA carriers such as extracellular vesicles (EVs) and Argonaute-2 (AGO2), making it a potential extracellular marker for hypoxia. Full article

Severe vascular and nerve damage from diabetes is a leading cause of erectile dysfunction (ED) and poor response to oral phosphodiesterase 5 inhibitors. Argonaute 2 (Ago2), a catalytic engine in mammalian RNA interference, is involved in neurovascular regeneration under inflammatory conditions. In the present study, we report that Ago2 administration can effectively improve penile erection by enhancing cavernous endothelial cell angiogenesis and survival under diabetic conditions. We found that although Ago2 is highly expressed around blood vessels and nerves, it is significantly reduced in the penis tissue of diabetic mice. Exogenous administration of the Ago2 protein restored erectile function in diabetic mice by reducing reactive oxygen species production-signaling pathways (inducing eNOS Ser¹¹⁷⁷/NF-κB Ser⁵³⁶ signaling) and improving cavernous endothelial angiogenesis, migration, and cell survival. Our study provides new evidence that Ago2 mediation may be a promising therapeutic strategy and a new approach for diabetic ED treatment. Full article

While host miRNA usually plays an antiviral role, the relentless tides of viral evolution have carved out a mechanism to recruit host miRNA as a viral protector. By complementing miR-122 at the 5′ end of the genome, the hepatitis C virus (HCV) gene can form a complex with Argonaute 2 (Ago2) protein to protect the 5′ end of HCV RNA from exonucleolytic attacks. Experiments showed that the disruption of the stem-loop 1(SL1) structure and the 9th nucleotide (T9) of HCV site 1 RNA could enhance the affinity of the Ago2 protein to the HCV site 1 RNA (target RNA). However, the underlying mechanism of how the conformation and dynamics of the Ago2: miRNA: target RNA complex is affected by the SL1 and T9 remains unclear. To address this, we performed large-scale molecular dynamics simulations on the AGO2-miRNA complex binding with the WT target, T9-abasic target and SL1-disruption target, respectively. The results revealed that the T9 and SL1 structures could induce the departing motion of the PAZ, PIWI and N domains, propping up the mouth of the central groove which accommodates the target RNA, causing the instability of the target RNA and disrupting the Ago2 binding. The coordinated motion among the PAZ, PIWI and N domains were also weakened by the T9 and SL1 structures. Moreover, we proposed a new model wherein the Ago2 protein could adopt a more constraint conformation with the proximity and more correlated motions of the PAZ, N and PIWI domains to protect the target RNA from dissociation. These findings reveal the mechanism of the Ago2-miRNA complex’s protective effect on the HCV genome at the atomic level, which will offer guidance for the design of drugs to confront the protection effect and engineering of Ago2 as a gene-regulation tool. Full article

Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections. Full article

As the most prominent member of the miR-17-92 cluster, miR-17-5p is well associated with tumorigenesis and cancer progression. It can exert both oncogenic and tumor-suppressive functions by inducing translational repression and/or mRNA decay. The complexity of the tissue-specific expression of the targeted transcripts seems to contribute to the differential functions of miR-17-5p in different types of cancers. In this study, we selected 12 reported miR-17-5p targeting genes with mRNA levels unaffected by miR-17-5p expression and analyzed their expression in 31 organ tissues in transgenic mice by real-time PCR. Surprisingly, miR-17-5p expressing transgenic mice showed a positive correlation in these tissues between miR-17-5p expression levels and the selected miR-17-5p targeted transcripts; with high expression of the miRNA in organs with high selected miRNA-targeted mRNA levels. In cancer cell lines, overexpression of 7 reported miR-17-5p targeted genes’ 3′-UTRs promoted miR-17-5p expression; meanwhile, transfection of 3′-UTRs with mutations had no significant effect. Moreover, an increase in AGO2 mRNA was associated with 3′-UTR expression as confirmed by real-time PCR. Hence, miR-17-5p regulation by these target genes might be an alternative mechanism to maintain miR-17-5p expression at tissue-specific levels. Full article

Long noncoding RNAs (lncRNAs) are involved in a variety of biological processes and illnesses. While a considerable number of lncRNAs have been discovered in skeletal muscle to far, their role and underlying processes during myogenesis remain mostly unclear. In this study, we described a new functional lncRNA named lncR-133a. Gene overexpression and interference studies in goat skeletal muscle satellite cells (MuSCs) were used to establish its function. The molecular mechanism by which lncR-133a governs muscle differentiation was elucidated primarily using quantitative real-time PCR (qRT-PCR), Western blotting, dual-luciferase activity assays, RNA immunoprecipitation, biotin-labeled probe, and RNA fluorescence in situ hybridization analyses. LncR-133a was found to be substantially expressed in longissimus thoracis et lumborum muscle, and its expression levels changed during MuSC differentiation in goats. We validated that lncR-133a suppresses MuSC differentiation in vitro. Dual-luciferase reporter screening, Argonaute 2 (AGO2) RNA immunoprecipitation assays, biotin-labeled lncR-133a capture, and fluorescence in situ hybridization showed that lncR-133a interacted with miR-133a-3p. Additionally, miR-133a-3p facilitated MuSC differentiation, but lncR-133a reversed this effect. The luciferase reporter assay and functional analyses established that miR-133a-3p directly targets fibroblast growth factor receptor 1 (FGFR1). Moreover, lncR-133a directly reduced miR-133a-3p’s capacity to suppress FGFR1 expression, and positively regulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). In summary, our results suggested that lncR-133a suppresses goat muscle differentiation by targeting miR-133a-3p and activating FGFR1/ERK1/2 signaling pathway. Full article

RNA-binding proteins (RBPs) play important roles in modulating miRNA-mediated mRNA target repression. Argonaute2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) that plays a central role in silencing mechanisms via small non-coding RNA molecules known as siRNAs and miRNAs. Small RNAs loaded into Argonaute proteins catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and mRNA target destabilization. In previous studies we have shown that KCC2, a neuronal Cl (−) extruding K (+) Cl (−) co-transporter 2, is regulated by miR-92 in neuronal cells. Searching for Ago2 partners by immunoprecipitation and LC-MS/MS analysis, we isolated among other proteins the Serpine mRNA binding protein 1 (SERBP1) from SH-SY5Y neuroblastoma cells. Exploring the role of SERBP1 in miRNA-mediated gene silencing in SH-SY5Y cells and primary hippocampal neurons, we demonstrated that SERBP1 silencing regulates KCC2 expression through the 3′ untranslated region (UTR). In addition, we found that SERBP1 as well as Ago2/miR-92 complex bind to KCC2 3′UTR. Finally, we demonstrated the attenuation of miR-92-mediated repression of KCC2 3′UTR by SERBP1 silencing. These findings advance our knowledge regarding the miR-92-mediated modulation of KCC2 translation in neuronal cells and highlight SERBP1 as a key component of this gene regulation. Full article