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Nature Jan 2024Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago...
Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA-target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA-DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a 'roadblock' that prevents the guide RNA 5' ends and the target DNA 3' ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA-DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.
Topics: Argonaute Proteins; Cryoelectron Microscopy; DNA; Enzyme Activation; NAD+ Nucleosidase; Nucleic Acid Conformation; Nucleic Acids; Protein Conformation; RNA, Guide, CRISPR-Cas Systems; Mutagenesis
PubMed: 37783228
DOI: 10.1038/s41586-023-06665-6 -
Cell Dec 2023MicroRNAs (miRNAs) guide Argonaute (AGO) proteins to bind and repress target RNAs. However, some unusual targets trigger destruction of the miRNA, a phenomenon known as...
MicroRNAs (miRNAs) guide Argonaute (AGO) proteins to bind and repress target RNAs. However, some unusual targets trigger destruction of the miRNA, a phenomenon known as target-directed miRNA degradation (TDMD). This Snapshot depicts our current understanding of how TDMD occurs and highlights established functions of TDMD in viruses and model organisms. To view this SnapShot, open or download the PDF.
Topics: MicroRNAs; Argonaute Proteins; RNA Stability
PubMed: 38065084
DOI: 10.1016/j.cell.2023.11.020 -
Cell Reports Aug 2023Biomolecular condensates have been shown to interact in vivo, yet it is unclear whether these interactions are functionally meaningful. Here, we demonstrate that...
Biomolecular condensates have been shown to interact in vivo, yet it is unclear whether these interactions are functionally meaningful. Here, we demonstrate that cooperativity between two distinct condensates-germ granules and P bodies-is required for transgenerational gene silencing in C. elegans. We find that P bodies form a coating around perinuclear germ granules and that P body components CGH-1/DDX6 and CAR-1/LSM14 are required for germ granules to organize into sub-compartments and concentrate small RNA silencing factors. Functionally, while the P body mutant cgh-1 is competent to initially trigger gene silencing, it is unable to propagate the silencing to subsequent generations. Mechanistically, we trace this loss of transgenerational silencing to defects in amplifying secondary small RNAs and the stability of WAGO-4 Argonaute, both known carriers of gene silencing memories. Together, these data reveal that cooperation between condensates results in an emergent capability of germ cells to establish heritable memory.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; RNA, Small Interfering; Gene Silencing; RNA Interference; Germ Cells; RNA Nucleotidyltransferases
PubMed: 37505984
DOI: 10.1016/j.celrep.2023.112859 -
Argonaute, Vault, and Ribosomal Proteins Targeted by Autoantibodies in Systemic Lupus Erythematosus.The Journal of Rheumatology Sep 2023To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations...
OBJECTIVE
To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations with serological and clinical variables.
METHODS
Human proteome arrays (> 21,000 proteins) were screened with serum from patients with SLE (n = 12) and healthy controls (n = 6) for IgG and IgA binding. Top hits were validated with 2 cohorts of patients with SLE (cohort 1, n = 49; cohort 2, n = 46) and other rheumatic diseases by ELISA. Clinical associations of the autoantibodies were tested.
RESULTS
Ro60 was the top hit in the screen, and the 10 following proteins included 2 additional known SLE autoantigens plus 8 novel autoantigens involved in microRNA processing (Argonaute protein 1 [AGO1], AGO2, and AGO3), ribosomes (ribosomal protein lateral stalk subunit P2 and ovarian tumor deubiquitinase 5 [OTUD5]), RNA transport by the vault (major vault protein), and the immune proteasome (proteasome activator complex subunit 3). Patient serum contained IgG reactive with these proteins and IgA against the AGO proteins. Using the 95th percentile of healthy donor reactivity, 5-43% were positive for the novel antigens, with OTUD5 and AGO1 showing the highest percentages of positivity. Autoantibodies against AGO1 proteins were more prevalent in patients with oral ulcers in a statistically significant manner. IgG autoantibodies against AGO proteins were also seen in other rheumatic diseases.
CONCLUSION
We discovered new autoantigens existing in cytosolic macromolecular protein assemblies containing RNA (except the proteasome) in cells. A more comprehensive list of autoantigens will allow for a better analysis of how proteins are targeted by the autoimmune response. Future research will also reveal whether specific autoantibodies have utility in the diagnosis or management of SLE.
Topics: Humans; Autoantibodies; Ribosomal Proteins; Proteasome Endopeptidase Complex; Argonaute Proteins; Lupus Erythematosus, Systemic; Autoantigens; Immunoglobulin G; Immunoglobulin A
PubMed: 37127324
DOI: 10.3899/jrheum.2022-1327 -
Frontiers in Microbiology 2023It is vital to diagnose pathogens quickly and effectively in the research and treatment of disease. Argonaute (Ago) proteins are recently discovered nucleases with... (Review)
Review
It is vital to diagnose pathogens quickly and effectively in the research and treatment of disease. Argonaute (Ago) proteins are recently discovered nucleases with nucleic acid shearing activity that exhibit specific recognition properties beyond CRISPR-Cas nucleases, which are highly researched but restricted PAM sequence recognition. Therefore, research on Ago protein-mediated nucleic acid detection technology has attracted significant attention from researchers in recent years. Using Ago proteins in developing nucleic acid detection platforms can enable efficient, convenient, and rapid nucleic acid detection and pathogen diagnosis, which is of great importance for human life and health and technological development. In this article, we introduce the structure and function of Argonaute proteins and discuss the latest advances in their use in nucleic acid detection.
PubMed: 37744931
DOI: 10.3389/fmicb.2023.1255716 -
EMBO Reports Nov 2023MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs...
MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases - casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing.
Topics: Animals; Humans; Argonaute Proteins; Caenorhabditis elegans Proteins; Casein Kinase I; MicroRNAs; RNA-Induced Silencing Complex; Caenorhabditis elegans; Gene Silencing; Serine; RNA-Binding Proteins
PubMed: 37712432
DOI: 10.15252/embr.202357250 -
Current Opinion in Microbiology Aug 2023Both eukaryotes and prokaryotes (archaea and bacteria) encode an arsenal of immune systems that protect the host against mobile genetic elements (MGEs) including... (Review)
Review
Both eukaryotes and prokaryotes (archaea and bacteria) encode an arsenal of immune systems that protect the host against mobile genetic elements (MGEs) including viruses, plasmids, and transposons. Whereas Argonaute proteins (Agos) are best known for post-transcriptional gene silencing in eukaryotes, in all domains of life, members from the highly diverse Argonaute protein family act as programmable immune systems. To this end, Agos are programmed with small single-stranded RNA or DNA guides to detect and silence complementary MGEs. Across and within the different domains of life, Agos function in distinct pathways and MGE detection can trigger various mechanisms that provide immunity. In this review, we delineate the diverse immune pathways and underlying mechanisms for both eukaryotic Argonautes (eAgos) and prokaryotic Argonautes (pAgos).
Topics: Argonaute Proteins; Prokaryotic Cells; Bacteria; Eukaryota; Archaea
PubMed: 37023508
DOI: 10.1016/j.mib.2023.102313 -
Nature Oct 2023Transposable elements are genomic parasites that expand within and spread between genomes. PIWI proteins control transposon activity, notably in the germline. These...
Transposable elements are genomic parasites that expand within and spread between genomes. PIWI proteins control transposon activity, notably in the germline. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5'-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5'-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (mG-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5'-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.
Topics: Animals; Argonaute Proteins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; DNA Transposable Elements; Endoribonucleases; Holoenzymes; Piwi-Interacting RNA; RNA Cap Analogs
PubMed: 37758951
DOI: 10.1038/s41586-023-06588-2 -
The Journal of Biological Chemistry Jan 2024Argonaute (AGO) proteins in all three domains of life form ribonucleoprotein or deoxyribonucleoprotein complexes by loading a guide RNA or DNA, respectively. Since all... (Review)
Review
Argonaute (AGO) proteins in all three domains of life form ribonucleoprotein or deoxyribonucleoprotein complexes by loading a guide RNA or DNA, respectively. Since all AGOs retain a PIWI domain that takes an RNase H fold, the ancestor was likely an endoribonuclease (i.e., a slicer). In animals, most miRNA-mediated gene silencing occurs slicer independently. However, the slicer activity of AGO is indispensable in specific events, such as development and differentiation, which are critical for vertebrates and thus cannot be replaced by the slicer-independent regulation. This review highlights the distinctions in catalytic activation mechanisms among slicing-competent AGOs, shedding light on the roles of two metal ions in target recognition and cleavage. The precision of the target specificity by the RNA-induced silencing complexes is reevaluated and redefined. The possible coevolutionary relationship between slicer-independent gene regulation and AGO-binding protein, GW182, is also explored. These discussions reveal that numerous captivating questions remain unanswered regarding the timing and manner in which AGOs employ their slicing activity.
Topics: Animals; Argonaute Proteins; Ribonucleases; RNA, Guide, CRISPR-Cas Systems; RNA, Small Interfering; RNA-Induced Silencing Complex
PubMed: 38029964
DOI: 10.1016/j.jbc.2023.105499 -
Biological Chemistry Oct 2023MicroRNA (miRNA)-guided gene silencing is a key regulatory process in various organisms and linked to many human diseases. MiRNAs are processed from precursor molecules... (Review)
Review
MicroRNA (miRNA)-guided gene silencing is a key regulatory process in various organisms and linked to many human diseases. MiRNAs are processed from precursor molecules and associate with Argonaute proteins to repress the expression of complementary target mRNAs. Excellent work by numerous labs has contributed to a detailed understanding of the mechanisms of miRNA function. However, miRNA effects have mostly been analyzed and viewed as isolated events and their natural environment as part of complex RNA-protein particles (RNPs) is often neglected. RNA binding proteins (RBPs) regulate key enzymes of the miRNA processing machinery and furthermore RBPs or readers of RNA modifications may modulate miRNA activity on mRNAs. Such proteins may function similarly to miRNAs and add their own contributions to the overall expression level of a particular gene. Therefore, post-transcriptional gene regulation might be more the sum of individual regulatory events and should be viewed as part of a dynamic and complex RNP world.
Topics: Humans; RNA Interference; MicroRNAs; Gene Expression Regulation; RNA-Binding Proteins; Argonaute Proteins
PubMed: 37739934
DOI: 10.1515/hsz-2023-0203