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Viruses Mar 2024As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including...
As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including microcephaly and other congenital malformations, resulting in fatal intrauterine death. Therefore, developing sensitive and specific methods for the early detection and accurate diagnosis of the ZIKV is essential for controlling its spread and mitigating its impact on public health. Herein, we set up a novel nucleic acid detection system based on Argonaute (Ago)-mediated nucleic acid detection, targeting the non-structural protein 5 (NS5) region of the ZIKV genome (abbreviated ZIKV-PAND). Without preamplification with the polymerase chain reaction (PCR), the minimum detection concentration (MDC) of ZIKV-PAND was about 10 nM. When introducing an amplification step, the MDC can be dramatically decreased to the aM level (8.3 aM), which is comparable to qRT-PCR assay (1.6 aM). In addition, the diagnostic findings from the analysis of simulated clinical samples or Zika virus samples using ZIKV-PAND show a complete agreement of 100% with qRT-PCR assays. This correlation can aid in the implementation of molecular testing for clinical diagnoses and the investigation of ZIKV infection on an epidemiological scale.
Topics: Zika Virus; Zika Virus Infection; Humans; Viral Nonstructural Proteins; Pyrococcus furiosus; Argonaute Proteins; Sensitivity and Specificity; RNA, Viral; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Genome, Viral
PubMed: 38675882
DOI: 10.3390/v16040539 -
Non-coding RNA Mar 2024Argonaute proteins, guided by small RNAs, play crucial roles in gene regulation and genome protection through RNA interference (RNAi)-related mechanisms. Ribosomal RNAs... (Review)
Review
Argonaute proteins, guided by small RNAs, play crucial roles in gene regulation and genome protection through RNA interference (RNAi)-related mechanisms. Ribosomal RNAs (rRNAs), encoded by repeated rDNA units, constitute the core of the ribosome being the most abundant cellular transcripts. rDNA clusters also serve as sources of small RNAs, which are loaded into Argonaute proteins and are able to regulate rDNA itself or affect other gene targets. In this review, we consider the impact of small RNA pathways, specifically siRNAs and piRNAs, on rRNA gene regulation. Data from diverse eukaryotic organisms suggest the potential involvement of small RNAs in various molecular processes related to the rDNA transcription and rRNA fate. Endogenous siRNAs are integral to the chromatin-based silencing of rDNA loci in plants and have been shown to repress rDNA transcription in animals. Small RNAs also play a role in maintaining the integrity of rDNA clusters and may function in the cellular response to rDNA damage. Studies on the impact of RNAi and small RNAs on rRNA provide vast opportunities for future exploration.
PubMed: 38668377
DOI: 10.3390/ncrna10020019 -
EMBO Reports May 2024Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a...
Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.
Topics: Argonaute Proteins; MicroRNAs; Caveolin 1; Humans; Neoplasm Metastasis; Cell Line, Tumor; Animals; Gene Expression Regulation, Neoplastic; Extracellular Vesicles; Mice; Protein Binding; Neoplasms; Sirtuin 2
PubMed: 38649663
DOI: 10.1038/s44319-024-00132-7 -
Chemical Science Apr 2024Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid-based biosensors. The...
Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid-based biosensors. The specific binding and cleavage of nucleic acids by pAgo proteins, which are crucial processes for their applications, are dependent on the presence of Mn bound in the pockets, as verified through X-ray crystallography. However, a comprehensive understanding of how dissociated Mn in the solvent affects the catalytic cycle, and its underlying regulatory role in this structure-function relationship, remains underdetermined. By combining experimental and computational methods, this study reveals that unbound Mn in solution enhances the flexibility of diverse pAgo proteins. This increase in flexibility through decreasing the number of hydrogen bonds, induced by Mn, leads to higher affinity for substrates, thus facilitating cleavage. More importantly, Mn-induced structural flexibility increases the mismatch tolerance between guide-target pairs by increasing the conformational states, thereby enhancing the cleavage of mismatches. Further simulations indicate that the enhanced flexibility in linkers triggers conformational changes in the PAZ domain for recognizing various lengths of nucleic acids. Additionally, Mn-induced dynamic alterations of the protein cause a conformational shift in the N domain and catalytic sites towards their functional form, resulting in a decreased energy penalty for target release and cleavage. These findings demonstrate that the dynamic conformations of pAgo proteins, resulting from the presence of the unbound Mn in solution, significantly promote the catalytic cycle of endonucleases and the tolerance of cleavage to mismatches. This flexibility enhancement mechanism serves as a general strategy employed by Ago proteins from diverse prokaryotes to accomplish their catalytic functions and provide useful information for Ago-based precise molecular diagnostics.
PubMed: 38638240
DOI: 10.1039/d3sc06221j -
EMBO Reports May 2024The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the...
The PIWI-interacting RNA (piRNA) pathway plays a crucial role in silencing transposons in the germline. piRNA-guided target cleavage by PIWI proteins triggers the biogenesis of new piRNAs from the cleaved RNA fragments. This process, known as the ping-pong cycle, is mediated by the two PIWI proteins, Siwi and BmAgo3, in silkworms. However, the detailed molecular mechanism of the ping-pong cycle remains largely unclear. Here, we show that Spindle-E (Spn-E), a putative ATP-dependent RNA helicase, is essential for BmAgo3-dependent production of Siwi-bound piRNAs in the ping-pong cycle and that this function of Spn-E requires its ATPase activity. Moreover, Spn-E acts to suppress homotypic Siwi-Siwi ping-pong, but this function of Spn-E is independent of its ATPase activity. These results highlight the dual role of Spn-E in facilitating proper heterotypic ping-pong in silkworms.
Topics: Bombyx; Animals; RNA, Small Interfering; Insect Proteins; Adenosine Triphosphatases; RNA Helicases; Argonaute Proteins; Piwi-Interacting RNA
PubMed: 38632376
DOI: 10.1038/s44319-024-00137-2 -
Oncology Reports Mar 2024It has been reported that PL2L60 proteins, a product of gene which might be activated by an intragenic promoter, could mediate a common pathway specifically for...
It has been reported that PL2L60 proteins, a product of gene which might be activated by an intragenic promoter, could mediate a common pathway specifically for tumorigenesis. In the present study, it was further identified by using western blot assay that the PL2L60 proteins could be degraded in cancer cells through a mechanism of selective autophagy in response to oxidative stress. The PL2L60 was downregulated in various types of cancer cells under the hypoxic condition independently of HIF‑1α, resulting in apoptosis of cancer cells. Inhibition of autophagy by small interfering RNA targeting of either Beclin‑1 () or Atg5 resulted in restoration of PL2L60 expression in hypoxic cancer cell. The hypoxic degradation of PL2L60 was also blocked by the attenuation of the autophagosome membrane protein Atg8/microtubule‑associated protein 1 light chain 3 (LC3) or autophagy cargo protein p62 expression. Surprisingly, Immunofluorescence analysis demonstrated that LC3 could be directly bound to PL2L60 and was required for the transport of PL2L60 from the nucleus to the cytoplasm for lysosomal flux under basal or activated autophagy in cancer cells. Moreover, flow cytometric analysis displayed that knocking down of PL2L60 mRNA but not PIWIL2 mRNA effectively inhibited cancer cell proliferation and promoted apoptosis of cancer cells. The similar results were obtained from tumorigenic experiment, in which PL2L60 downregulation in necroptosis areas was confirmed by immunohistochemistry. These results suggested that various cancer could be suppressed by promoting autophagy. The present study revealed a key role of autophagic degradation of PL2L60 in hypoxia‑induced cancer cell death, which could be used as a novel therapeutic target of cancer.
Topics: Humans; RNA, Small Interfering; Neoplasms; Hypoxia; Apoptosis; Autophagy; Stress, Physiological; RNA, Messenger; Argonaute Proteins
PubMed: 38624021
DOI: 10.3892/or.2024.8700 -
Nucleic Acids Research Jun 2024Eukaryotic Argonaut proteins (AGOs) assemble RNA-induced silencing complexes (RISCs) with guide RNAs that allow binding to complementary RNA sequences and subsequent...
Eukaryotic Argonaut proteins (AGOs) assemble RNA-induced silencing complexes (RISCs) with guide RNAs that allow binding to complementary RNA sequences and subsequent silencing of target genes. The model plant Arabidopsis thaliana encodes 10 different AGOs, categorized into three distinct clades based on amino acid sequence similarity. While clade 1 and 2 RISCs are known for their roles in post-transcriptional gene silencing, and clade 3 RISCs are associated with transcriptional gene silencing in the nucleus, the specific mechanisms of how RISCs from each clade recognize their targets remain unclear. In this study, I conducted quantitative binding analyses between RISCs and target nucleic acids with mismatches at various positions, unveiling distinct target binding characteristics unique to each clade. Clade 1 and 2 RISCs require base pairing not only in the seed region but also in the 3' supplementary region for stable target RNA binding, with clade 1 exhibiting a higher stringency. Conversely, clade 3 RISCs tolerate dinucleotide mismatches beyond the seed region. Strikingly, they bind to DNA targets with an affinity equal to or surpassing that of RNA, like prokaryotic AGO complexes. These insights challenge existing views on plant RNA silencing and open avenues for exploring new functions of eukaryotic AGOs.
Topics: Arabidopsis; Arabidopsis Proteins; RNA-Induced Silencing Complex; Argonaute Proteins; RNA, Plant; Protein Binding; RNA Interference; Base Pair Mismatch; DNA, Plant
PubMed: 38621714
DOI: 10.1093/nar/gkae257 -
Nucleic Acids Research Jun 2024MicroRNAs (miRNAs) are essential regulators of several biological processes. They are loaded onto Argonaute (AGO) proteins to achieve their repressive function, forming...
MicroRNAs (miRNAs) are essential regulators of several biological processes. They are loaded onto Argonaute (AGO) proteins to achieve their repressive function, forming the microRNA-Induced Silencing Complex known as miRISC. While several AGO proteins are expressed in plants and animals, it is still unclear why specific AGOs are strictly binding miRNAs. Here, we identified the co-chaperone DNJ-12 as a new interactor of ALG-1, one of the two major miRNA-specific AGOs in Caenorhabditis elegans. DNJ-12 does not interact with ALG-2, the other major miRNA-specific AGO, and PRG-1 and RDE-1, two AGOs involved in other small RNA pathways, making it a specific actor in ALG-1-dependent miRNA-mediated gene silencing. The loss of DNJ-12 causes developmental defects associated with defective miRNA function. Using the Auxin Inducible Degron system, a powerful tool to acutely degrade proteins in specific tissues, we show that DNJ-12 depletion hampers ALG-1 interaction with HSP70, a chaperone required for miRISC loading in vitro. Moreover, DNJ-12 depletion leads to the decrease of several miRNAs and prevents their loading onto ALG-1. This study uncovers the importance of a co-chaperone for the miRNA function in vivo and provides insights to explain how different small RNAs associate with specific AGO in animals.
Topics: Animals; Argonaute Proteins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; HSP70 Heat-Shock Proteins; MicroRNAs; Molecular Chaperones; Protein Binding; RNA-Binding Proteins; HSP40 Heat-Shock Proteins
PubMed: 38613392
DOI: 10.1093/nar/gkae272 -
Nature Communications Apr 2024Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays...
Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We show that upregulation of ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms' ability to withstand oxidative stress.
Topics: Animals; Humans; Caenorhabditis elegans; Caenorhabditis elegans Proteins; MicroRNAs; Oxidative Stress; Glucagon-Like Peptide 1; RNA-Binding Proteins
PubMed: 38594249
DOI: 10.1038/s41467-024-47306-4 -
Frontiers in Plant Science 2024RNA-directed DNA methylation (RdDM) is driven by small RNAs (sRNAs) complementary to the nascent transcript of RNA polymerase V (Pol V). sRNAs associated with ARGONAUTE...
RNA-directed DNA methylation (RdDM) is driven by small RNAs (sRNAs) complementary to the nascent transcript of RNA polymerase V (Pol V). sRNAs associated with ARGONAUTE (AGO) proteins are tethered to Pol V mainly by the AGO-hook domain of its subunit NRPE1. We found, by analyses, that Pol V strongly colocalizes on chromatin with another AGO-hook protein, SPT6-like (SPT6L), which is a known essential transcription elongation factor of Pol II. Our phylogenetic analysis revealed that SPT6L acquired its AGO-binding capacity already in the most basal streptophyte algae, even before the emergence of Pol V, suggesting that SPT6L might be a driving force behind the RdDM evolution. Since its emergence, SPT6L with the AGO-hook represents the only conserved SPT6 homolog in , implying that the same protein is involved in both Pol II and Pol V complexes. To better understand the role of SPT6L in the Pol V complex, we characterized genomic loci where these two colocalize and uncovered that DNA methylation there is more dynamic, driven by higher levels of sRNAs often from non-canonical RdDM pathways and more dependent on chromatin modifying and remodeling proteins like MORC. Pol V loci with SPT6L are highly depleted in helitrons but enriched in gene promoters for which locally and temporally precise methylation is necessary. In view of these results, we discuss potential roles of multiple AGO-hook domains present in the Pol V complex and speculate that SPT6L mediates methylation of naïve loci by interconnecting Pol II and Pol V activities.
PubMed: 38576781
DOI: 10.3389/fpls.2024.1372880