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Gene Apr 2019microRNAs (miRNAs) play important role in the regulation of placental development, and abnormal miRNA expression is associated with preeclampsia (PE). miRNAs are...
INTRODUCTION
microRNAs (miRNAs) play important role in the regulation of placental development, and abnormal miRNA expression is associated with preeclampsia (PE). miRNAs are released from trophoblast cells to maternal blood flow, where they are highly stable, being encapsulated inside extracellular vesicles, like exosomes or bound to Argonaute proteins. In PE, placental dysfunction leads to aberrant extracellular miRNA secretion. hsa-miR-210 is a hypoxia-sensitive miRNA found to be upregulated in PE; however, it is unknown whether it is the cause or the consequence of the disease.
OBJECTIVE
Our aim was to analyze the expression of several miRNAs, including hsa-miR-210 in placenta, exosome and Ago-bound fractions comparing normal (N) and PE pregnancies. We performed in vitro analyses of extracellular hsa-miR-210 secretion of trophoblast cell cultures (of villous and extravillous origin) under hypoxic condition.
METHODS
PE and N placenta samples were collected from C-sections, and blood samples were drawn from each pregnant woman in the third trimester. HTR-8 and JAR cell lines were cultured in exosome-free media and treated with hypoxia-mimetic agents. Exosome and Ago-bound fractions were isolated by membrane affinity spin column method from plasma and cell media. Short RNAs were extracted from exosomes and vesicle-free fractions, and total-RNA was isolated from the placenta samples. The RNA purity and concentration were measured by spectrophotometry. Expression analysis was carried out by qPCR with specific primers to target and reference miRNAs.
RESULTS
The level of hsa-miR-210 was significantly higher in PE placentas, which could cause a minor increase of exosomal and a high elevation of Ago-bound miR-210 in circulation. Hypoxia lead to intracellular hsa-miR-210 upregulation in trophoblast cell lines. In extravillous cell (HTR-8) media, only the level of exosomal hsa-miR-210 was increased but no change in Ago-bound hsa-miR-210 level was observed. In contrast, in villous cell (JAR) media, the level of exosomal hsa-miR-210 was increased and enhanced release of Ago-bound hsa-miR-210 was also observed.
CONCLUSION
Based on our data, we postulate that in PE, exosomal hsa-miR-210 is secreted actively from the trophoblast, and by intercellular communication, it may have a role in disease etiology. In addition, there is a passive release of Ago-bound hsa-miR-210 into the circulation, which may represent by-products of cell-death and is thereby a possible consequence of the disease.
Topics: Adult; Argonaute Proteins; Cell Hypoxia; Exosomes; Female; Gene Expression Regulation, Developmental; Humans; MicroRNAs; Placenta; Pre-Eclampsia; Pregnancy; Trophoblasts
PubMed: 30659946
DOI: 10.1016/j.gene.2019.01.012 -
Molecular Cell Oct 2014Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for gametogenesis, embryogenesis, and stem cell maintenance in animals. Piwi proteins act on transposon... (Review)
Review
Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for gametogenesis, embryogenesis, and stem cell maintenance in animals. Piwi proteins act on transposon RNAs by cleaving the RNAs and by interacting with factors involved in RNA regulation. Additionally, piRNAs generated from transposons and psuedogenes can be used by Piwi proteins to regulate mRNAs at the posttranscriptional level. Here we discuss piRNA biogenesis, recent findings on posttranscriptional regulation of mRNAs by the piRNA pathway, and the potential importance of this posttranscriptional regulation for a variety of biological processes such as gametogenesis, developmental transitions, and sex determination.
Topics: Argonaute Proteins; Gene Expression Regulation; Models, Genetic; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; RNA, Small Interfering; Sex Determination Processes
PubMed: 25280102
DOI: 10.1016/j.molcel.2014.09.012 -
RNA Biology 2018Viruses masterfully regulate host gene expression during infection. Many do so, in part, by expressing non-coding RNAs. Recent work has shown that HSUR 2, a viral... (Review)
Review
Viruses masterfully regulate host gene expression during infection. Many do so, in part, by expressing non-coding RNAs. Recent work has shown that HSUR 2, a viral non-coding RNA expressed by the oncogenic Herpesvirus saimiri, regulates mRNA expression through a novel mechanism. HSUR 2 base pairs with both target mRNAs and host miRNAs in infected cells. This results in HSUR 2-dependent recruitment of host miRNAs and associated Ago proteins to target mRNAs, and the subsequent destabilization of target mRNAs. Using this mechanism, this virus regulates key cellular pathways during viral infection. Here I discuss the evolution of our thinking about HSUR function and explore the implications of recent findings in relation to the current views on the functions of interactions between miRNAs and other classes of non-coding RNAs, the potential advantages of this mechanism of regulation of gene expression, and the evolutionary origin of HSUR 2.
Topics: Argonaute Proteins; Base Sequence; Gene Expression Regulation, Viral; Herpesviridae Infections; Herpesvirus 2, Saimiriine; Host-Pathogen Interactions; Humans; RNA, Messenger; RNA, Untranslated; RNA, Viral
PubMed: 29895222
DOI: 10.1080/15476286.2018.1467176 -
MBio Dec 2018Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic...
Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition-and sometimes cleavage-of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and revealed their striking diversity in comparison with eAgos. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage. Many pAgos are associated with putative nucleases, helicases, and DNA binding proteins in the same gene or operon, suggesting that they are involved in target processing. The great variability of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and for their use as potential tools in genome editing. The eukaryotic Ago proteins and the RNA interference pathways they are involved in are widely used as a powerful tool in research and as potential therapeutics. In contrast, the properties and functions of prokaryotic Ago (pAgo) proteins have remained poorly understood. Understanding the diversity and functions of pAgos holds a huge potential for discovery of new cellular pathways and novel tools for genome manipulations. Only few pAgos have been characterized by structural or biochemical approaches, while previous genomic studies discovered about 300 proteins in archaeal and eubacterial genomes. Since that time the number of bacterial strains with sequenced genomes has greatly expanded, and many previously sequenced genomes have been revised. We undertook comprehensive analysis of pAgo proteins in sequenced genomes and almost tripled the number of known genes of this family. Our research thus forms a foundation for further experimental characterization of pAgo functions that will be important for understanding of the basic biology of these proteins and their adoption as a potential tool for genome engineering in the future.
Topics: Archaea; Archaeal Proteins; Argonaute Proteins; Bacteria; Bacterial Proteins; Eukaryota; Gene Editing; Gene Transfer, Horizontal; Genome; Protein Binding; RNA Interference
PubMed: 30563906
DOI: 10.1128/mBio.01935-18 -
Nucleic Acids Research May 2023Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium...
Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium butyricum, induces DNA interference between homologous sequences and generates double-stranded breaks (DSBs) in target DNAs. This mechanism enables the host to defend against invading DNAs such as plasmids and viruses. However, whether such a CbAgo-mediated DNA cleavage is mutagenic remains unexplored. Here we demonstrate that CbAgo, directed by plasmid-encoded guide sequences, can cleave genome target sites and induce chromosome recombination between downstream homologous sequences in Escherichia coli. The recombination rate correlates well with pAgo DNA cleavage activity and the mechanistic study suggests the recombination involves DSBs and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. We anticipate this novel DNA-guided interference to be useful in broader genetic manipulation. Our study also provides an in vivo assay to characterize or engineer pAgo DNA cleavage activity.
Topics: Argonaute Proteins; DNA; Escherichia coli; Plasmids; Prokaryotic Cells; Sequence Homology; Genome, Bacterial
PubMed: 36928676
DOI: 10.1093/nar/gkad188 -
Biochemical Society Transactions Aug 2013In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and... (Review)
Review
In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism.
Topics: Animals; Argonaute Proteins; Caenorhabditis elegans
PubMed: 23863149
DOI: 10.1042/BST20130086 -
Cell Reports Oct 2022Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans...
Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5'-terminus nucleotides of 5'P guides, but no obvious preferences for that in 5'OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3'-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5'OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques.
Topics: Argonaute Proteins; RNA; DNA, Single-Stranded; RNA Cleavage; DNA; Nucleotides; RNA, Guide, CRISPR-Cas Systems
PubMed: 36288702
DOI: 10.1016/j.celrep.2022.111533 -
Seminars in Cell & Developmental Biology May 2017MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the... (Review)
Review
MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss recent single-molecule and high-resolution structural data in the light of theoretical work appropriated from the study of transcription-factor search. We suggest that experimentally observed internal states of the Ago-miRNA search complex may have the explicit biological function of speeding up search while maintaining specificity.
Topics: Argonaute Proteins; Binding Sites; Diffusion; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Humans; MicroRNAs; Protein Binding; Protein Conformation; RNA Stability; RNA, Messenger; Signal Transduction; Thermodynamics
PubMed: 27235676
DOI: 10.1016/j.semcdb.2016.05.017 -
Nature Communications Jan 2024Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In...
Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In contrast, the functions of prokaryotic counterparts (pAgo) are less well known. Recently, short pAgos in conjunction with the associated TIR or Sir2 (SPARTA or SPARSA) were found to serve as antiviral systems to combat phage infections. Herein, we present the cryo-EM structures of nicotinamide adenine dinucleotide (NAD)-bound SPARSA with and without nucleic acids at resolutions of 3.1 Å and 3.6 Å, respectively. Our results reveal that the APAZ (Analogue of PAZ) domain and the short pAgo form a featured architecture similar to the long pAgo to accommodate nucleic acids. We further identified the key residues for NAD binding and elucidated the structural basis for guide RNA and target DNA recognition. Using structural comparisons, molecular dynamics simulations, and biochemical experiments, we proposed a putative mechanism for NAD hydrolysis in which an H186 loop mediates nucleophilic attack by catalytic water molecules. Overall, our study provides mechanistic insight into the antiphage role of the SPARSA system.
Topics: NAD; RNA, Guide, CRISPR-Cas Systems; Argonaute Proteins; Bacteriophages; Nucleic Acids
PubMed: 38200015
DOI: 10.1038/s41467-023-44660-7 -
BMC Plant Biology Jan 2015Argonaute (AGO) proteins form the core of the RNA-induced silencing complex, a central component of the smRNA machinery. Although reported from several plant species,...
BACKGROUND
Argonaute (AGO) proteins form the core of the RNA-induced silencing complex, a central component of the smRNA machinery. Although reported from several plant species, little is known about their evolution. Moreover, these genes have not yet been cloned from the ecological model plant, Nicotiana attenuata, in which the smRNA machinery is known to mediate important ecological traits.
RESULTS
Here, we not only identify 11 AGOs in N. attenuata, we further annotate 133 genes in 17 plant species, previously not annotated in the Phytozome database, to increase the number of plant AGOs to 263 genes from 37 plant species. We report the phylogenetic classification, expansion, and diversification of AGOs in the plant kingdom, which resulted in the following hypothesis about their evolutionary history: an ancestral AGO underwent duplication events after the divergence of unicellular green algae, giving rise to four major classes with subsequent gains/losses during the radiation of higher plants, resulting in the large number of extant AGOs. Class-specific signatures in the RNA-binding and catalytic domains, which may contribute to the functional diversity of plant AGOs, as well as context-dependent changes in sequence and domain architecture that may have consequences for gene function were found.
CONCLUSIONS
Together, the results demonstrate that the evolution of AGOs has been a dynamic process producing the signatures of functional diversification in the smRNA pathways of higher plants.
Topics: Amino Acid Sequence; Argonaute Proteins; Evolution, Molecular; Molecular Sequence Data; Phylogeny; Plant Proteins; Plants; Protein Structure, Tertiary; Sequence Alignment; Nicotiana
PubMed: 25626325
DOI: 10.1186/s12870-014-0364-6