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Overall folding topology of Argonaute family. a Linear representation of the domain architecture of eAgo and pAgo (long pAgo, short pAgo and PIWI-RE). b hAgo2 (PDB:4F3T) structure (a cartoon representation on the left and a surface representation on the right) with N (green), Linker L1 (gray), PAZ (blue), Linker L2 (pale cyan), MID (orange), and PIWI (purple) domains along with its linear representation with residues numbered in the upper left, (c). As in b but for MjAgo and d as in b but for AfAgo. hAgo2 and AfAgo bound to a guide RNA colored in wheat. hAgo2 indicated as eAgo, MjAgo indicated as long pAgo, and the AfAgo indicated as short pAgo
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Argonaute proteins are highly conserved and widely expressed in almost all organisms. They not only play a critical role in the biogenesis of small RNAs but also defend against invading nucleic acids via small RNA or DNA-mediated gene silencing pathways. One functional mechanism of Argonaute proteins is acting as a nucleic-acid-guided endonuclease,...
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Prokaryotic Argonaute proteins (pAgos) widely participate in hosts to defend against the invasion of nucleic acids. Compared with the CRISPR-Cas system , which requires a specific motif on the target and can only use RNA as guide, pAgos exhibit precise endonuclease activity on any arbitrary target sequence and can use both RNA and DNA as guide, thu...
Citations
... eAgos and long pAgos share a bilobed structural architecture ( 17 ). The MID-PIWI lobe consists of the middle (MID) and p-element induced wimpy testis (PIWI) domains ( 18 ), and is responsible for guide 5 end binding and guide-mediated target binding (18)(19)(20)(21). This lobe is connected through the linker 2 (L2) motif to the N-terminal lobe, which is comprised of the N-terminal (N) domain, the linker 1 (L1) motif and the PAZ (PIWI-ARGONAUTE-ZWILLE) domain. ...
... eAgos and long pAgos share a bilobed structural architecture ( 17 ). The MID-PIWI lobe consists of the middle (MID) and p-element induced wimpy testis (PIWI) domains ( 18 ), and is responsible for guide 5 end binding and guide-mediated target binding (18)(19)(20)(21). This lobe is connected through the linker 2 (L2) motif to the N-terminal lobe, which is comprised of the N-terminal (N) domain, the linker 1 (L1) motif and the PAZ (PIWI-ARGONAUTE-ZWILLE) domain. ...
In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.
... The N-terminal domain is variable and essential for driving duplex sRNAs unwinding and loading a guide strand to assemble mature RISCs (Kwak and Tomari 2012), whereas C-terminal PAZ, MID, and PIWI domains are conserved (Tolia and Joshua-Tor 2007;Hutvagner and Simard 2008). These domains collaboratively use sRNAs to bind and mediate the cleavage of complementary RNA targets (Jin et al. 2021). The PAZ domain contains a binding pocket and is responsible for binding the 3′-end of sRNAs (Yan et al. 2003;Lingel et al. 2004;Ma et al. 2004;Tian et al. 2011). ...
... The MID domain affords an insertion site for the 5′-end of sRNAs (Boland et al. 2011;Frank et al. 2012). The PIWI domain has functions similar to an RNase H and contains a conserved active site and strongly implicates AGOs in sRNA-directed mRNA cleavage or slicer activity (Liu et al. 2004;Song et al. 2004;Jin et al. 2021). Different AGO proteins bind to different-sized or competitively bind to sRNAs to perform diverse functions (Martin-Merchan et al. 2023), although their typical functions are in cleavage and translational inhibition of target mRNAs to repress the expression of targeted genes in the cytoplasm (Ma and Zhang 2018;Feng et al. 2021). ...
Argonaute (AGO) proteins are highly conserved and widely distributed across various organisms. They mainly associate with small RNAs (sRNAs) and act as integral players of the RNA-induced silencing complex in the RNA interference (RNAi) pathway, regulating gene expression at transcriptional and post-transcriptional levels, thereby mounting diverse fine-tuning functions in a variety of biological processes. Since the discovery and functional characterization of the first AGO in Arabidopsis, our understanding of the functions of AGO proteins has grown rapidly throughout the plant kingdom. AGO1 attracts investigators’ attention because it forms an autoregulatory loop with miR168 and associates with other endogenous sRNAs and cross-kingdom exogenous sRNAs to relay all-round functions. AGO1 associates with endogenous sRNAs that form a complicated regulatory network via targeting a large body of downstream genes involved in growth, development, and stress-induced responses. Host AGO1 may also be exploited by cross-kingdom exogenous sRNAs generated by parasitic organisms to facilitate their colonization via suppressing host defense genes. Moreover, many pathogenic microbes directly target host AGO1 to facilitate their infection via suppression of the host RNAi pathway. Thus, we focus on plant AGO1 and provide an overview of our current understanding of the roles of AGO1 in the coordination of plant disease resistance with growth and development. We also discuss the perspectives in the dissection of the AGO1-mediated signal transduction pathway.
... The first AGO protein was discovered 25 years ago by Bohmert et al. (1998) in Arabidopsis, and the name derived from the octopus-like phenotype of ago1 Arabidopsis mutant plants (Bohmert et al., 1998). Within a few years, AGO proteins had been described in most kinds of eukaryotes, being involved in RNA interference (Hammond et al., 2001;Vaucheret et al., 2004;Jin et al., 2021). Eukaryotic AGO proteins can be phylogenetically divided into three different subfamilies: the AGO subfamily, analogous to Arabidopsis AGO1, the PIWI subfamily, analogous to Drosophila melanogaster P-element induced wimpy testis (PIWI) (only present in metazoans), and the worm-specific AGO cluster (WAGO) subfamily (Fig. 1A). ...
... Eukaryotic AGO proteins can be phylogenetically divided into three different subfamilies: the AGO subfamily, analogous to Arabidopsis AGO1, the PIWI subfamily, analogous to Drosophila melanogaster P-element induced wimpy testis (PIWI) (only present in metazoans), and the worm-specific AGO cluster (WAGO) subfamily (Fig. 1A). AGO proteins are not only widespread in eukaryotes, they have also been found in ~30% of archaea and ~10% of bacteria, including Pyrococcus furiosus, Marinitoga piezophile, and Thermus thermophiles (Jin et al., 2021). ...
ARGONAUTE (AGO) proteins are the final effectors of small RNA-mediated transcriptional and post-transcriptional silencing pathways. Plant AGO proteins are essential preserving genome integrity, regulating developmental processes, and during stress responses and pathogen defence. Since the discovery of the first eukaryotic AGO in Arabidopsis thaliana, our understanding of these proteins has grown exponentially throughout all eukaryotes. However, many aspects of AGO proteins modes of action and how these are influenced by their subcellular localizations have remained to be elucidated. Here, we provide an updated and comprehensive view of the evolution, domain architecture/roles, expression pattern, subcellular localization, and biological functions of the ten AGO proteins in Arabidopsis.
... Argonaute proteins are part of the RNA-induced-silencing-complex (RISC), which plays a central role shaping the transcriptome through RNA interference [50]. After binding to mRNA, Ago2 cuts the complementary strand via an endonuclease activity into single-stranded siRNA/miRNA [51]. ...
LIM and SH3 protein 1 was originally identified as a structural cytoskeletal protein with scaffolding function. However, recent data suggest additional roles in cell signaling and gene expression, especially in tumor cells. These novel functions are primarily regulated by the site-specific phosphorylation of LASP1. This review will focus on specific phosphorylation-dependent interaction between LASP1 and cellular proteins that orchestrate primary tumor progression and metastasis. More specifically, we will describe the role of LASP1 in chemokine receptor, and PI3K/AKT signaling. We outline the nuclear role for LASP1 in terms of epigenetics and transcriptional regulation and modulation of oncogenic mRNA translation. Finally, newly identified roles for the cytoskeletal function of LASP1 next to its known canonical F-actin binding properties are included.
... From the structural and mechanistic point of view, all characterised eAgos are very similar, as they all use small (~ 21-30 nt) RNA molecules as guides for sequence-speci c recognition of RNA targets, and are monomeric proteins sharing four conserved functional domains, which are organized in a bilobed structure [3]. The N-terminal lobe consists of N-domain that separates guide and target strands [4], and PAZ domain responsible for binding the 3′-terminus of the guide RNA; the C-terminal lobe consists of MID domain, which binds the 5′-terminus of the guide RNA, and PIWI domain, a nuclease [1,2,5,6]. Upon recognition of the RNA target, eAgos may either cleave it employing catalytic activity of the PIWI domain or, particularly eAgo proteins that encode catalytically inactive PIWI domains, recruit partner proteins leading to degradation of the target RNA or repression of its translation [1,7]. Target speci city of eAgos is determined solely by correct base pairing between guide and target RNA strands. ...
Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like ‘long’ and truncated ‘short’ pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a short Argonaute protein encoded by an archaeon Archaeoglobus fulgidus . We demonstrate that AfAgo associates with small RNA molecules carrying 5′-terminal AUU nucleotides in vivo , and characterize its affinity to various RNA and DNA guide/target strands in vitro . We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.
... eAgo proteins, as the core component of the RNA-induced silencing complex, have become a key player in RNA interference (RNAi) pathways (3). The eAgo proteins mainly form binary complexes with their guide RNAs and recognize complementary target mRNAs for subsequent site-specific cleavage or silencing, which then leads to the degradation and inhibition of protein translation (4). However, due to lack of RNAi pathways in archaea and bacteria, the physiological function of prokaryotic Ago (pAgo) proteins has remained elusive for a long time (5). ...
... PIWI-RE proteins with unknown functions appear in several major bacterial lineages, and their domain constituent is similar to short pAgo proteins with two conserved MID and PIWI domains (19). But unlike short pAgo proteins, PIWI-RE proteins have two conserved residues: arginine (R) and glutamate (E) in PIWI and MID domains, which are essential for nucleic acid-binding (4,19). Currently, although the function and mechanism of eAgo and several long pAgo proteins have been studied in detail, little is known about those of short pAgo and PIWI-RE proteins (20)(21)(22)(23). ...
Argonaute (Ago) proteins are widely expressed in almost all organisms. Eukaryotic Ago (eAgo) proteins bind small RNA guides forming RNA-induced silencing complex that silence gene expression, and prokaryotic Ago (pAgo) proteins defend against invading nucleic acids via binding small RNAs or DNAs. pAgo proteins have shown great potential as a candidate ‘scissors’ for gene editing. Protein domains are fundamental units of protein structure, function and evolution; however, the domains of Ago proteins are not well annotated/curated currently. Therefore, full functional domain annotation of Ago proteins is urgently needed for researchers to understand the function and mechanism of Ago proteins. Herein, we constructed the first comprehensive domain annotation database of Ago proteins (AGODB). The database curates detailed information of 1902 Ago proteins, including 1095 eAgos and 807 pAgos. Especially for long pAgo proteins, all six domains are annotated and curated. Gene Ontology (GO) enrichment analysis revealed that Ago genes in different species were enriched in the following GO terms: biological processes (BPs), molecular function and cellular compartment. GO enrichment analysis results were integrated into AGODB, which provided insights into the BP that Ago genes may participate in. AGODB also allows users to search the database with a variety of options and download the search results. We believe that the AGODB will be a useful resource for understanding the function and domain components of Ago proteins. This database is expected to cater to the needs of scientific community dedicated to the research of Ago proteins.
Database URL
http://i.uestc.edu.cn/agodb/
... Analiza funkcjonalnych domen eukariotycznych AGO wyłoniła trzy podrodziny: AGO, PIWI i WAGO (ang. WORM-SPECIFIC ARGONAUTE) [6] . Białka PIWI i oddziałujące z nimi sRNA (tzw. ...
Białka ARGONAUT (AGO) są integralnymi elementami regulatorowych szlaków znajdujących się pod kontrolą małych RNA (ang. small RNA, sRNA), o fundamentalnym
znaczeniu dla właściwego funkcjonowania komórek eukariotycznych. AGO, jako wysoce
wyspecjalizowane platformy wiążące specyficzne sRNA, koordynują wyciszanie genów poprzez interakcję z innymi czynnikami białkowymi (tworząc tzw. kompleks RISC, ang. RNA--induced silencing complex), przyczyniając się do endonukleolitycznego cięcia docelowego mRNA i/lub wpływając na proces translacji. Coraz więcej dowodów potwierdza również udział białek AGO w kilku innych procesach komórkowych, takich jak np.: regulacja transkrypcji, sekwestracja, zależna od RNA metylacja DNA, naprawa uszkodzeń DNA, synteza siRNA niezależna od białek DCL (ang. DICER-like), czy też kotranskrypcyjna regulacja ekspresji genów MIRNA i splicingu intronów. Poszczególne gatunki roślin charakteryzują się obecnością różnej liczby białek AGO, w wielu przypadkach o nieznanej jeszcze regulatorowej i/lub biologicznej funkcji. Niniejszy artykuł przeglądowy obejmuje aktualną wiedzę na temat funkcji roślinnych AGO w biologii komórki i rozwoju roślin.
... AGO proteins are core components of the RNA-induced silencing complex (RISC), which is part of RNA interference (RNAi) processing. They bind guide RNAs (siRNA, miRNA) and process them for subsequent site-specific cleavage for the silencing of target mRNAs involved in development, differentiation, and protection against viral infection [147]. AGO2 consists of four domains: the N-terminal, PAZ, Mid, and PIWI domains. ...
DNA and RNA binding proteins (DRBPs) are a broad class of molecules that regulate numerous cellular processes across all living organisms, creating intricate dynamic multilevel networks to control nucleotide metabolism and gene expression. These interactions are highly regulated, and dysregulation contributes to the development of a variety of diseases, including cancer. An increasing number of proteins with DNA and/or RNA binding activities have been identified in recent years, and it is important to understand how their activities are related to the molecular mechanisms of cancer. In addition, many of these proteins have overlapping functions, and it is therefore essential to analyze not only the loss of function of individual factors, but also to group abnormalities into specific types of activities in regard to particular cancer types. In this review, we summarize the classes of DNA-binding, RNA-binding, and DRBPs, drawing particular attention to the similarities and differences between these protein classes. We also perform a cross-search analysis of relevant protein databases, together with our own pipeline, to identify DRBPs involved in cancer. We discuss the most common DRBPs and how they are related to specific cancers, reviewing their biochemical, molecular biological, and cellular properties to highlight their functions and potential as targets for treatment.
... The conserved PAZ domain contains a specific binding pocket that can recognize and bind the 3' ends of small RNAs, which is essential for protecting small RNAs from degradation (Hutvagner and Simard 2008;Wu et al. 2020). The PIWI domain shows extensive homology to RNase H, which contains a slicer active site that cleaves target RNA complementary to small RNA (Jin et al. 2021). In previous studies, AGO7 was highly selective for interaction with miR390, which controls the cleavage of TAS3 precursor RNA to trigger the generation of tasiR-ARFs (Montgomery et al. 2008). ...
Key message
A melon gene MSO1 located on chromosome 10 by map-based cloning strategy, which encodes an ARGONAUTE 7 protein, is responsible for the development of shoot organization.
Abstract
Plant endogenous small RNAs (sRNAs) are involved in various plant developmental processes. In Arabidopsis, sRNAs combined with ARGONAUTE (AGO) proteins are incorporated into the RNA-induced silencing complex (RISC), which functions in RNA silencing or biogenesis of trans-acting siRNAs (ta-siRNAs). However, their roles in melon (Cucumis melo L.) are still unclear. Here, the melon shoot organization 1 (mso1) mutant was identified and shown to exhibit pleiotropic phenotypes in leaf morphology and plant architecture. Positional cloning of MSO1 revealed that it encodes a homologue of ArabidopsisAGO7/ZIPPY, which is required for the production of ta-siRNAs. The AG-to-C mutation in the second exon of MSO1 caused a frameshift mutation and significantly reduced its expression. Ectopic expression of MSO1 rescued the Arabidopsisago7 phenotype. RNA-seq analysis showed that several genes involved in transcriptional regulation and plant hormones were significantly altered in mso1 compared to WT. A total of 304 and 231 miRNAs were identified in mso1 and WT by sRNA sequencing, respectively, and among them, 42 known and ten novel miRNAs were differentially expressed. cme-miR390a significantly accumulated, and the expression levels of the two ta-siRNAs were almost completely abolished in mso1. Correspondingly, their targets, the ARF3 and ARF4 genes, showed dramatically upregulated expression, indicating that the miR390-TAS3-ARF pathway has conserved roles in melon. These findings will help us better understand the molecular mechanisms of MSO1 in plant development in melon.
... As another programmable nuclease with diverse binding and cleavage activities, pAgos are being exploited in genome editing applications (9), molecular cloning (24), and nucleic acid detection (25)(26)(27)(28). To date, catalytically active pAgos characterized have been isolated from thermophilic and mesophilic species (29). Interestingly, pAgos from mesophilic species analyzed so far are active in a wide temperature range from 37 • C to 50 • C (17,22), suggesting the possibility of finding pAgos cleaving targets with a high efficiency at 37 • C also in psychrotolerant or psychrophilic species. ...
Argonaute (Ago) proteins are programmable nucleases found in eukaryotes and prokaryotes. Prokaryotic Agos (pAgos) share a high degree of structural homology with eukaryotic Agos (eAgos), and eAgos originate from pAgos. Although eAgos exclusively cleave RNA targets, most characterized pAgos cleave DNA targets. This study characterized a novel pAgo, MbpAgo, from the psychrotolerant bacterium Mucilaginibacter paludis which prefers to cleave RNA targets rather than DNA targets. Compared to previously studied Agos, MbpAgo can utilize both 5'phosphorylated(5'P) and 5'hydroxylated(5'OH) DNA guides (gDNAs) to efficiently cleave RNA targets at the canonical cleavage site if the guide is between 15 and 17 nt long. Furthermore, MbpAgo is active at a wide range of temperatures (4-65°C) and displays no obvious preference for the 5'-nucleotide of a guide. Single-nucleotide and most dinucleotide mismatches have no or little effects on cleavage efficiency, except for dinucleotide mismatches at positions 11-13 that dramatically reduce target cleavage. MbpAgo can efficiently cleave highly structured RNA targets using both 5'P and 5'OH gDNAs in the presence of Mg2+ or Mn2+. The biochemical characterization of MbpAgo paves the way for its use in RNA manipulations such as nucleic acid detection and clearance of RNA viruses.
