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Phillip D Zamore - One of the best experts on this subject based on the ideXlab platform.
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to degrade a microrna destroy its Argonaute Protein
Molecular Cell, 2021Co-Authors: Phillip D ZamoreAbstract:Han et al. (2020) and Shi et al. (2020) report that the E3 ubiquitin ligase ZSWIM8 senses when an RNA and an Argonaute Protein-bound microRNA are extensively base paired and directs Argonaute destruction by the proteasome. The result is degradation of the microRNA.
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a dna guided Argonaute Protein functions in dna replication in thermus thermophilus
bioRxiv, 2019Co-Authors: Samson M Jolly, Ildar Gainetdinov, Lara Strittmatter, Avantika Dhabaria, Gregory M Hendricks, Beatrix Ueberheide, Phillip D ZamoreAbstract:Abstract Argonaute Proteins use nucleic acid guides to protect organisms against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. TtAgo binds small DNA guides derived from the chromosomal region where replication terminates and associates with Proteins known to act in DNA replication. T. thermophilus deploys a single type II topoisomerase, gyrase. When gyrase is inhibited, T. thermophilus relies on TtAgo to complete replication of its circular genome; loss of both gyrase and TtAgo activity produces long filaments that fail to separate into individual bacteria. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes made by DNA replication. One Sentence Summary The DNA-guided Argonaute Protein of Thermus thermophilus helps separate daughter chromosomes at the end of DNA replication.
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a dna guided Argonaute Protein functions in dna replication in thermus thermophilus preprint
bioRxiv, 2019Co-Authors: Samson M Jolly, Ildar Gainetdinov, Lara Strittmatter, Avantika Dhabaria, Gregory M Hendricks, Beatrix Ueberheide, Han Zhang, Phillip D ZamoreAbstract:Abstract Argonaute Proteins use nucleic acid guides to protect organisms against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. TtAgo binds small DNA guides derived from the chromosomal region where replication terminates and associates with Proteins known to act in DNA replication. T. thermophilus deploys a single type II topoisomerase, gyrase. When gyrase is inhibited, T. thermophilus relies on TtAgo to complete replication of its circular genome; loss of both gyrase and TtAgo activity produces long filaments that fail to separate into individual bacteria. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes made by DNA replication. One Sentence Summary The DNA-guided Argonaute Protein of Thermus thermophilus helps separate daughter chromosomes at the end of DNA replication.
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an automated bayesian pipeline for rapid analysis of single molecule binding data
bioRxiv, 2018Co-Authors: Carlas Smith, Samson M Jolly, Phillip D Zamore, Karina Jouravleva, Maximiliaan Huisman, David GrunwaldAbstract:Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute Protein from the Eubacterium Thermus thermophilus. We also used the pipeline to extend our understanding of TtAgo by measuring the Protein's binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.
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pitfalls of mapping high throughput sequencing data to repetitive sequences piwi s genomic targets still not identified
Developmental Cell, 2015Co-Authors: Georgi K Marinov, Phillip D Zamore, Alexei A. Aravin, Jie Wang, Dominik Handler, Barbara J Wold, Julius Brennecke, Katalin Fejes TothAbstract:Huang et al. (2013) recently reported that chromatin immunoprecipitation sequencing (ChIP-seq) reveals the genome-wide sites of occupancy by Piwi, a piRNA-guided Argonaute Protein central to transposon silencing in Drosophila. Their study also reported that loss of Piwi causes widespread rewiring of transcriptional patterns, as evidenced by changes in RNA polymerase II occupancy across the genome. Here we reanalyze their data and report that the underlying deep-sequencing dataset does not support the authors' genome-wide conclusions.
Gunter Meister - One of the best experts on this subject based on the ideXlab platform.
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peptide based isolation of Argonaute Protein complexes using ago app
Methods of Molecular Biology, 2017Co-Authors: Judith Hauptmann, Gunter MeisterAbstract:Argonaute (Ago) Proteins bind small RNAs such as microRNAs (miRNAs) or short interfering RNAs (siRNAs), which guide them to distinct mRNAs for post-transcriptional gene silencing. Mammalian miRNA-guided gene silencing pathways mainly lead to translational repression and mRNA destabilization. To facilitate these processes, Ago Proteins bind members of the GW Protein family, which form central interaction platforms for the recruitment of downstream effector Proteins. GW Proteins use tryptophane residues (W) to bind to the surface of Ago Proteins. This high affinity interaction is retained when a short, GST-fused GW peptide is used in biochemical pull-down experiments-an approach referred to as "Ago Affinity Purification by Peptides" (Ago-APP). Since the binding interface is conserved among different paralogues and different species, Ago-APP represents a universal tool to purify Ago Proteins and associated small RNAs using samples from species with conserved miRNA pathways.
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assembly and function of small rna Argonaute Protein complexes
Biological Chemistry, 2014Co-Authors: Anne Dueck, Gunter MeisterAbstract:Small RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs) or Piwi-interacting RNAs (piRNAs) are important regulators of gene expression in various organisms. Small RNAs bind to a member of the Argonaute Protein family and are incorporated into larger structures that mediate diverse gene silencing events. The loading of Argonaute Proteins with small RNAs is aided by a number of auxiliary factors as well as ATP hydrolysis. This review will focus on the mechanisms of Argonaute loading in different organisms. Furthermore, we highlight the versatile functions of small RNA-Argonaute Protein complexes in organisms from all three kingdoms of life.
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an Argonaute Protein directs nuclear xrn2 function
Molecular Cell, 2012Co-Authors: Daniele Hasler, Gunter MeisterAbstract:Argonaute Proteins are the mediators of small RNA-guided gene silencing pathways. In this issue, Couvillion and coworkers (Couvillion et al., 2012) found an unexpected function for a Tetrahymena Argonaute Protein: It forms a complex with tRNA fragments and is required for nuclear Xrn2 localization and function.
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experimental identification of microrna targets by immunoprecipitation of Argonaute Protein complexes
Methods of Molecular Biology, 2011Co-Authors: Michaela Beitzinger, Gunter MeisterAbstract:MicroRNAs (miRNAs) represent a class of small noncoding RNAs that negatively regulate gene expression-. Intensive research during the past decade has established miRNAs as key regulators of many cellular pathways. MiRNAs have also been implicated in a number of diseases including various forms of cancer. Mammalian miRNAs associate with members of the Argonaute (Ago) Protein family and function in multi-Protein complexes. MiRNAs guide Ago Protein complexes to partially complementary sequences typically located in the 3' untranslated region (UTR) of their target mRNAs leading to the inhibition of its translation and/or its destabilization. To understand the biological roles of miRNAs, it is essential to identify the mRNA targets that they regulate. Because of the low degree of complementarity between the miRNA and its target sequence, it is often difficult to find targets computationally. Therefore, biochemical methods are needed to identify miRNA targets experimentally. The availability of highly specific monoclonal antibodies against Argonaute Proteins allows for the isolation of functional Ago-miRNA-mRNA complexes from -different cell lines, tissues, or even patient samples. Here we provide a detailed protocol for isolation and identification of miRNA target mRNAs from immunoprecipitated human Ago Protein complexes.
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the Argonaute Protein family
Genome Biology, 2008Co-Authors: Julia Hock, Gunter MeisterAbstract:Argonaute Proteins were first discovered genetically, and extensive research in the past few years has revealed that members of the Argonaute Protein family are key players in gene-silencing pathways guided by small RNAs. Small RNAs such as short interfering RNAs (siRNAs), microRNAs (miRNAs) or Piwi-interacting RNAs (piRNAs) are anchored into specific binding pockets and guide Argonaute Proteins to target mRNA molecules for silencing or destruction. Various classes of small RNAs and Argonaute Proteins are found in all higher eukaryotes and have important functions in processes as diverse as embryonic development, cell differentiation and transposon silencing. Argonaute Proteins are evolutionarily conserved and can be phylogenetically subdivided into the Ago subfamily and the Piwi subfamily. Ago Proteins are ubiquitously expressed and bind to siRNAs or miRNAs to guide post-transcriptional gene silencing either by destabilization of the mRNA or by translational repression. The expression of Piwi Proteins is mostly restricted to the germ line and Piwi Proteins associate with piRNAs to facilitate silencing of mobile genetic elements. Although various aspects of Argonaute function have been identified, many Argonaute Proteins are still poorly characterized. Therefore, it is very likely that as yet unknown functions of the Argonaute Protein family will be elucidated in the future.
Mikiko C. Siomi - One of the best experts on this subject based on the ideXlab platform.
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piwi interacting rna in drosophila biogenesis transposon regulation and beyond
Chemical Reviews, 2017Co-Authors: Haruna Yamashiro, Mikiko C. SiomiAbstract:PIWI-interacting RNAs (piRNAs) are germline-enriched small RNAs that control transposons to maintain genome integrity. To achieve this, upon being processed from piRNA precursors, most of which are transcripts of intergenic piRNA clusters, piRNAs bind PIWI Proteins, germline-specific Argonaute Proteins, to form effector complexes. The mechanism of this piRNA-mediated transposon silencing pathway is fundamentally similar to that of siRNA/miRNA-dependent gene silencing in that a small RNA guides its partner Argonaute Protein to target gene transcripts for repression via RNA–RNA base pairing. However, the uniqueness of this piRNA pathway has emerged through intensive genetic, biochemical, bioinformatic, and structural investigations. Here, we review the studies that elucidated the piRNA pathway, mainly in Drosophila, by describing both historical and recent progress. Studies in other species that have made important contributions to the field are also described.
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biogenesis of small rnas in animals
Nature Reviews Molecular Cell Biology, 2009Co-Authors: Mikiko C. SiomiAbstract:Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs in eukaryotic cells. Various small RNAs can be classified into three classes based on their biogenesis mechanism and the type of Argonaute Protein that they are associated with. Small RNAs of 20–30 nucleotides can target both chromatin and transcripts, and thereby keep both the genome and the transcriptome under extensive surveillance. Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs in eukaryotic cells. Various small RNAs of distinctive characteristics have been found and can be classified into three classes based on their biogenesis mechanism and the type of Argonaute Protein that they are associated with: microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs or esiRNAs) and Piwi-interacting RNAs (piRNAs). This Review summarizes our current knowledge of how these intriguing molecules are generated in animal cells.
Ghosh S. - One of the best experts on this subject based on the ideXlab platform.
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Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation.
'EMBO', 2020Co-Authors: Ghosh S., Guimaraes J. C), Lanzafame M., Schmidt A., Syed A. Pasha), Dimitriades B., Börsch A., Mittal N., Montavon T.Abstract:Translational readthrough, i.e., elongation of polypeptide chains beyond the stop codon, was initially reported for viral RNA, but later found also on eukaryotic transcripts, resulting in proteome diversification and Protein-level modulation. Here, we report that AGO1x, an evolutionarily conserved translational readthrough isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double-stranded RNAs (dsRNAs) and consequent induction of interferon responses and apoptosis. In contrast to other mammalian Argonaute Protein family members with primarily cytoplasmic functions, AGO1x exhibits nuclear localization in the vicinity of nucleoli. We identify AGO1x interaction with the polyribonucleotide nucleotidyltransferase 1 (PNPT1) and show that the depletion of this Protein further augments dsRNA accumulation. Our study thus uncovers a novel function of an Argonaute Protein in buffering the endogenous dsRNA-induced interferon responses, different than the canonical function of AGO Proteins in the miRNA effector pathway. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study thus suggests a new direction for limiting tumor growth.journal article2020 Aug 192020 08 19importe
Hiroaki Tabara - One of the best experts on this subject based on the ideXlab platform.
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in vitro analyses of the production and activity of secondary small interfering rnas in c elegans
The EMBO Journal, 2007Co-Authors: Kazuma Aoki, Hiromi Moriguchi, Tomoko Yoshioka, Katsuya Okawa, Hiroaki TabaraAbstract:In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) play important roles as intermediates. Primary siRNAs are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer; in some organisms, secondary siRNAs are also produced by processes involving RNA-dependent RNA polymerases (RdRPs), which act on target mRNAs. Using a cell-free assay system prepared from Caenorhabditis elegans, we analyzed the production and activity of secondary siRNAs. In this cell-free system, RdRP activity acts on mRNA-derived templates to produce small RNAs. The RRF-1 complex is predominantly responsible for the RdRP activity, and synthesizes secondary-type siRNA molecules in a Dicer-independent manner. Notably, secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute Protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Secondary rather than primary siRNAs may play a major role in the destabilization of target transcripts during RNAi in C. elegans.