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Frontiers in Bioengineering and... 2024Boolean gates, the fundamental components of digital circuits, have been widely investigated in synthetic biology because they permit the fabrication of biosensors and...
Boolean gates, the fundamental components of digital circuits, have been widely investigated in synthetic biology because they permit the fabrication of biosensors and facilitate biocomputing. This study was conducted to design and construct Boolean gates in the yeast , the main component of which was the RNA interference pathway (RNAi) that is naturally absent from the budding yeast cells. We tested different expression cassettes for the siRNA precursor (a giant hairpin sequence, a DNA fragment-flanked by one or two introns-between convergent promoters or transcribed separately in the sense and antisense directions) and placed different components under the control of the circuit inputs (i.e., the siRNA precursor or proteins such as the Dicer and the Argonaute). We found that RNAi-based logic gates are highly sensitive to promoter leakage and, for this reason, challenging to implement . Convergent-promoter architecture turned out to be the most reliable solution, even though the overall best performance was achieved with the most difficult design based on the siRNA precursor as a giant hairpin.
PubMed: 38895554
DOI: 10.3389/fbioe.2024.1392967 -
International Journal of Molecular... May 2024DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific...
DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific gene expression during spermatogenesis of Japanese flounder (), the expression profiles of () and () genes in the gonads of female, male, and sex-reversed pseudo-male were analyzed, and the dynamic of DNA methylation was investigated. As a result, and genes were highly expressed in the testis of both male and pseudo-male , with significant variation among male individuals. The DNA methylation levels in the promoter regions of both and were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of genes during spermatogenesis. There was also sperm quality variation among male , and the sperm curvilinear velocity was positively correlated with the expression of both and genes. These results indicated that the DNA methylation in and promoter regions may affect the initiation of gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in .
Topics: Animals; Male; DNA Methylation; Argonaute Proteins; Flounder; Spermatozoa; Spermatogenesis; Female; Promoter Regions, Genetic; Testis; Gene Expression Regulation; Fish Proteins
PubMed: 38892123
DOI: 10.3390/ijms25115935 -
Scientific Data Jun 2024In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in...
In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a consequence of cell confluency, AGO2 protein translocates into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.
Topics: Humans; Argonaute Proteins; Transcriptome; Proteomics; MicroRNAs; HEK293 Cells; Cell Line, Tumor; Gene Expression Profiling
PubMed: 38866801
DOI: 10.1038/s41597-024-03465-z -
Nature Communications Jun 2024A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P) hydrolysis, was recently identified...
A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P) hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15-3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection.
Topics: Argonaute Proteins; Cryoelectron Microscopy; Crystallography, X-Ray; Bacterial Proteins; Protein Domains; DNA, Single-Stranded; RNA, Guide, CRISPR-Cas Systems; Models, Molecular; Nucleic Acids; Protein Binding
PubMed: 38844755
DOI: 10.1038/s41467-024-49271-4 -
Viruses Apr 2024The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing...
The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In , if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.
Topics: Argonaute Proteins; Cucumovirus; Arabidopsis; Arabidopsis Proteins; Protein Binding; Viral Proteins; Host-Pathogen Interactions; Viral Replicase Complex Proteins; Plant Diseases; RNA-Dependent RNA Polymerase; Methyltransferases
PubMed: 38793558
DOI: 10.3390/v16050676 -
Biosensors May 2024Since SARS-CoV-2 is a highly transmissible virus, alternative reliable, fast, and cost-effective methods are still needed to prevent virus spread that can be applied in...
Since SARS-CoV-2 is a highly transmissible virus, alternative reliable, fast, and cost-effective methods are still needed to prevent virus spread that can be applied in the laboratory and for point-of-care testing. Reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) is currently the gold criteria for detecting RNA viruses, which requires reverse transcriptase to reverse transcribe viral RNA into cDNA, and fluorescence quantitative PCR detection was subsequently performed. The frequently used reverse transcriptase is thermolabile; the detection process is composed of two steps: the reverse transcription reaction at a relatively low temperature, and the qPCR performed at a relatively high temperature, moreover, the RNA to be detected needs to pretreated if they had advanced structure. Here, we develop a fast and sensitive one-tube SARS-CoV-2 detection platform based on Ultra-fast RTX-PCR and Argonaute-mediated Nucleic acid Detection (PAND) technology (URPAND). URPAND was achieved ultra-fast RTX-PCR process based on a thermostable RTX (exo-) with both reverse transcriptase and DNA polymerase activity. The URPAND can be completed RT-PCR and PAND to detect nucleic acid in one tube within 30 min. This method can specifically detect SARS-CoV-2 with a low detection limit of 100 copies/mL. The diagnostic results of clinical samples with one-tube URPAND displayed 100% consistence with RT-qPCR test. Moreover, URPAND was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The URPAND platform is rapid, accurate, tube closed, one-tube, easy-to-operate and free of large instruments, which provides a new strategy to the detection of SARS-CoV-2 and other RNA viruses.
Topics: SARS-CoV-2; Pyrococcus furiosus; RNA, Viral; COVID-19; Humans; Argonaute Proteins; Real-Time Polymerase Chain Reaction; Biosensing Techniques; COVID-19 Nucleic Acid Testing
PubMed: 38785719
DOI: 10.3390/bios14050245 -
Characterization of Argonaute-containing protein complexes in Leishmania-infected human macrophages.PloS One 2024The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide....
The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide. Unfortunately, due to the unavailability of approved vaccines for humans and the limited efficacy of available drugs, leishmaniasis is on the rise. A comprehensive understanding of host-pathogen interactions at the molecular level could pave the way to counter leishmaniasis. There is growing evidence that several intracellular pathogens target RNA interference (RNAi) pathways in host cells to facilitate their persistence. The core elements of the RNAi system are complexes of Argonaute (Ago) proteins with small non-coding RNAs, also known as RNA-induced silencing complexes (RISCs). Recently, we have shown that Leishmania modulates Ago1 protein of host macrophages for its survival. In this study, we biochemically characterize the Ago proteins' interactome in Leishmania-infected macrophages compared to non-infected cells. For this, a quantitative proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC) was employed, followed by purification of host Ago-complexes using a short TNRC6 protein-derived peptide fused to glutathione S-transferase beads as an affinity matrix. Proteomic-based detailed biochemical analysis revealed Leishmania modulated host macrophage RISC composition during infection. This analysis identified 51 Ago-interacting proteins with a broad range of biological activities. Strikingly, Leishmania proteins were detected as part of host Ago-containing complexes in infected cells. Our results present the first report of comprehensive quantitative proteomics of Ago-containing complexes isolated from Leishmania-infected macrophages and suggest targeting the effector complex of host RNAi machinery. Additionally, these results expand knowledge of RISC in the context of host-pathogen interactions in parasitology in general.
Topics: Argonaute Proteins; Humans; Macrophages; Proteomics; Leishmania; RNA Interference; Leishmaniasis
PubMed: 38781128
DOI: 10.1371/journal.pone.0303686 -
PloS One 2024Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there...
Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.
Topics: Animals; Female; Male; Mice; Argonaute Proteins; CRISPR-Cas Systems; Mice, Inbred C57BL; Mice, Transgenic; Pseudopregnancy
PubMed: 38771805
DOI: 10.1371/journal.pone.0296414 -
BioRxiv : the Preprint Server For... May 2024Mammalian pericentromeric tandem repeats produce long noncoding RNAs (lncRNAs) that are dysregulated in cancer and linked to genomic instability. Identifying the basic...
Mammalian pericentromeric tandem repeats produce long noncoding RNAs (lncRNAs) that are dysregulated in cancer and linked to genomic instability. Identifying the basic molecular characteristics of these lncRNAs and their regulation is important to understanding their biological function. Here, we determine that the Argonaute (Ago) proteins of the RNA interference (RNAi) pathway directly and uniformly repress bidirectional pericentromeric lncRNAs in a Dicer-dependent manner in mouse embryonic and adult stem cells. Ago-dependent and Dicer-dependent autoregulatory small RNAs were identified within pericentromeric lncRNA degradation intermediates. We develop an RNase H cleavage assay to determine the relative proportions and lengths of the pericentromeric lncRNA targets. We find that 5'-phosphate and non-polyadenylated bidirectional pericentromeric lncRNAs are expressed at similar proportions. These lncRNAs can span up to 9 repeats, with transcription from the reverse strand template yielding the longer products. Using pericentromeric repeat RNA reporters, we determine that Ago represses pericentromeric lncRNAs after S phase transcription. Upon loss of Ago, pericentromeric lncRNA dysregulation results in delayed cell cycle progression, a defective mitotic spindle assembly checkpoint (SAC) and genomic instability. These results show that an evolutionarily conserved Ago activity at pericentromeres contributes to mammalian genome stability.
PubMed: 38765997
DOI: 10.1101/2024.05.09.593425 -
Nature Communications May 2024Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may...
Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may result in their elimination. Chromosomal integration of ARGs preserves resistance advantage while relieving the selective pressure for keeping costly plasmids. In some bacterial lineages, such as carbapenemase producing sequence type ST38 Escherichia coli, most ARGs are chromosomally integrated. Here we reproduce by experimental evolution the mobilisation of the carbapenemase bla gene from the pOXA-48 plasmid into the chromosome. We demonstrate that this integration depends on a plasmid-induced fitness cost, a mobile genetic structure embedding the ARG and a novel antiplasmid system ApsAB actively involved in pOXA-48 destabilization. We show that ApsAB targets high and low-copy number plasmids. ApsAB combines a nuclease/helicase protein and a novel type of Argonaute-like protein. It belongs to a family of defense systems broadly distributed among bacteria, which might have a strong ecological impact on plasmid diffusion.
Topics: Plasmids; beta-Lactamases; Escherichia coli; Escherichia coli Proteins; Bacterial Proteins; Anti-Bacterial Agents; Drug Resistance, Bacterial; Chromosomes, Bacterial
PubMed: 38750030
DOI: 10.1038/s41467-024-48219-y