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1
Heimstadt, Susanne Barbara. "Functional diversification of Arabidopsis Argonaute proteins." Thesis, University of East Anglia, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492957.
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Yigit, Erbay. "The Argonaute Family of Genes in Caenorhabditis Elegans: a Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/328.
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Members of the Argonaute family of proteins, which interact with small RNAs, are the key players of RNAi and other related pathways. The C. elegans genome encodes 27 members of the Argonaute family. During this thesis research, we sought to understand the functions of the members of this gene family in C. elegans. Among the Argonaute family members, rde-1 and alg-1/2have previously been shown to be essential for RNAi and development, respectively. In this work, we wanted to assign functions to the remaining members of this large family of proteins.
Here, we describe the phenotype of 31 deletion alleles representing all of the previously uncharacterized Argonaute members. In addition to rde-1, our analysis revealed that two other Argonaute members csr-1 and prg-1 are also essential for development. csr-1 is partially required for RNAi, and essential for proper chromosome segregation. prg-1, a member of PIWI subfamily of Argonaute genes, exhibits reduced brood size and temperature-sensitive sterile phenotype, implicating that it is required for germline maintenance.
Additionally, we showed that RDE-1 interacts with trigger-derived sense and antisense siRNAs (primary siRNAs) to initiate RNAi, while several other Argonaute proteins, SAGO-1, SAGO-2, and perhaps others, functioning redundantly, interact with amplified siRNAs (secondary siRNAs) to mediate downstream silencing. Moreover, our analysis uncovered that another member of Argonaute gene family, ergo-1, is essential for the endogenous RNAi pathway.
Furthermore, we built an eight-fold Argonaute mutant, MAGO8, and analyzed its developmental phenotype and sensitivity to RNAi. Our analysis revealed that the genes deleted in the MAGO8 mutant function redundantly with each other, and are required for RNAi and the maintenance of the stem cell totipotency.
3
Åström, Miranda. "Search for the Argonaute protein that governs miRNA regulation in Dictyostelium discoideum." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-432967.
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MicroRNAs are small non-coding RNAs that regulate gene expression through RNA interference. These small RNAs enact gene silencing by forming a RNA-inducing silencing complex together with the effector protein Argonaute. The function of the Argonautes in the social amoeba Dictyostelium discoideum is not yet fully understood. In this study, we look closer at Argonaute B by investigating if it is possible to extract the protein from the cells by the addition of a polypeptide protein tag called 3xFlag. At the same time, we also look into if Argonaute B is important for cell growth. Sequences of the 3xFlag tag with or without the Argonaute B gene (agnB) attached had previously been cloned into a vector and transformed into Dictyostelium discoideum cell. The 3xFlag::agnB sequence was confirmed in wild type and agnB knock-out strains through polymerase chain reaction. We then verified the expression of the fusion protein in the cells by western blot. The cell growth was measured by how the number of cells changed over time. The experiment suggested that Argonaute B is important for growth. Our result show that the construct 3xFlag::agnB sequenced had correctly been transformed into the strains and is highly expressed under tested conditions. We could also see that Argonaute B is an important factor in cell growth.
4
Seth, Meetu. "Functions of Argonaute Proteins in Self Versus Non-Self Recognition in the C. elegans Germline: A Dissertation." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/874.
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Organisms employ sophisticated mechanisms to silence foreign nucleic acid, such as viruses and transposons. Evidence exists for pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), but the mechanisms that distinguish “self” from “non-self” are not well understood. Our studies on transgene silencing in C. elegans have uncovered an RNA surveillance system in which the PIWI protein, PRG-1, uses a vast repertoire of piRNAs to recognize foreign transcripts and to initiate epigenetic silencing. Partial base pairing by piRNAs is sufficient to guide PRG-1 targeting. PRG-1 in turn recruits RdRP to synthesize perfectly matching antisense siRNAs (22G-RNAs) that are loaded onto worm-specific Argonaute (WAGO) proteins. WAGOs collaborate with chromatin factors to maintain epigenetic silencing (RNAe). Since mismatches are allowed during piRNA targeting, piRNAs could—in theory— target any transcript expressed in the germline, but germline genes are not subject to silencing by RNAe. Moreover, some foreign sequences are expressed and appear to be adopted as “self.” How are “self” transcripts distinguished from foreign transcripts? We have found that another Argonaute, CSR-1, and its siRNAs—also synthesized by RdRP—protect endogenous genes from silencing by RNAe. We refer to this pathway as RNA-mediated gene activation (RNAa). Reducing CSR-1 or PRG-1 or increasing piRNA targeting can shift the balance towards expression or silencing, indicating that PRG-1 and CSR-1 compete for control over their targets. Thus worms have evolved a remarkable nucleic acids immunity mechanism in which opposing Argonaute pathways generate and maintain epigenetic memories of self and non-self nucleotide sequences.
5
Seth, Meetu. "Functions of Argonaute Proteins in Self Versus Non-Self Recognition in the C. elegans Germline: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/874.
Full textAbstract:
Organisms employ sophisticated mechanisms to silence foreign nucleic acid, such as viruses and transposons. Evidence exists for pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), but the mechanisms that distinguish “self” from “non-self” are not well understood. Our studies on transgene silencing in C. elegans have uncovered an RNA surveillance system in which the PIWI protein, PRG-1, uses a vast repertoire of piRNAs to recognize foreign transcripts and to initiate epigenetic silencing. Partial base pairing by piRNAs is sufficient to guide PRG-1 targeting. PRG-1 in turn recruits RdRP to synthesize perfectly matching antisense siRNAs (22G-RNAs) that are loaded onto worm-specific Argonaute (WAGO) proteins. WAGOs collaborate with chromatin factors to maintain epigenetic silencing (RNAe). Since mismatches are allowed during piRNA targeting, piRNAs could—in theory— target any transcript expressed in the germline, but germline genes are not subject to silencing by RNAe. Moreover, some foreign sequences are expressed and appear to be adopted as “self.” How are “self” transcripts distinguished from foreign transcripts? We have found that another Argonaute, CSR-1, and its siRNAs—also synthesized by RdRP—protect endogenous genes from silencing by RNAe. We refer to this pathway as RNA-mediated gene activation (RNAa). Reducing CSR-1 or PRG-1 or increasing piRNA targeting can shift the balance towards expression or silencing, indicating that PRG-1 and CSR-1 compete for control over their targets. Thus worms have evolved a remarkable nucleic acids immunity mechanism in which opposing Argonaute pathways generate and maintain epigenetic memories of self and non-self nucleotide sequences.
6
Salomon, William E. "Single-Molecule Imaging Reveals that Argonaute Re-Shapes the Properties of its Nucleic Acid Guides: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/804.
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Small RNA silencing pathways regulate development, viral defense, and genomic integrity in all kingdoms of life. An Argonaute (Ago) protein, guided by a tightly bound, small RNA or DNA, lies at the core of these pathways. Argonaute uses its small RNA or DNA to find its target sequences, which it either cleaves or stably binds, acting as a binding scaffold for other proteins. We used Co-localization Single-Molecule Spectroscopy (CoSMoS) to analyze target binding and cleavage by Ago and its guide. We find that both eukaryotic and prokaryotic Argonaute proteins re-shape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization: a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Counter to the rules of nucleic acid hybridization alone, we find that mouse AGO2 and its guide bind to microRNA targets 17,000 times tighter than the guide without Argonaute. Moreover, AGO2 can distinguish between microRNA-like targets that make seven base pairs with the guide and the products of cleavage, which bind via nine base pairs: AGO2 leaves the cleavage products faster, even though they pair more extensively.
This thesis presents a detailed kinetic interrogation of microRNA and RNA interference pathways. We discovered sub-domains within the previously defined functional domains created by Argonaute and its bound DNA or RNA guide. These sub-domains have features that no longer conform to the well-established properties of unbound oligonucleotides. It is by re-writing the rules for nucleic acid hybridization that Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than that of RNA or DNA. Taken altogether, these studies further our understanding about the biology of small RNA silencing pathways and may serve to guide future work related to all RNA-guided endonucleases.
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Urban, Marc [Verfasser], and Thomas [Akademischer Betreuer] Dresselhaus. "Gametogenesis-related small RNAs and Argonaute Proteins in Arabidopsis thaliana / Marc Urban ; Betreuer: Thomas Dresselhaus." Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1136471480/34.
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Zeitler, Daniela [Verfasser], and Gunter [Akademischer Betreuer] Meister. "Regulation of human Argonaute proteins and its implications in disease / Daniela Zeitler ; Betreuer: Gunter Meister." Regensburg : Universitätsbibliothek Regensburg, 2020. http://d-nb.info/1210701936/34.
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Urban, Marc Verfasser], and Thomas [Akademischer Betreuer] [Dresselhaus. "Gametogenesis-related small RNAs and Argonaute Proteins in Arabidopsis thaliana / Marc Urban ; Betreuer: Thomas Dresselhaus." Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1136471480/34.
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Jacobsen, Annette. "Lactobacilli Suppress Gene Expression of Key Proteins Involved in miRNA Biogenesis in HT29 and VK2/E6E7 Cells." Thesis, Örebro universitet, Institutionen för naturvetenskap och teknik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-32633.
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It has previously been demonstrated that lactic acid bacteria are able to influence the innate immune response of host cells. One way this can be achieved is through modulation of inflammatory cascades initiated by pattern recognition elements such as toll-like receptors. Micro RNA can also have an effect on innate immunity, and has been shown to have an influence in regulation of these pathways in immune responsive cells. However, it is yet to be determined if the interaction between lactic acid bacteria and host cells involves regulation of the RNA interference machinery involved in micro RNA biogenesis. Three of the key proteins responsible for miRNA production and activation are Argonaute 2, Dicer and Drosha. Together, these are responsible for the processing and activation of miRNA to enable post-transcriptional gene regulation. In this study we have used quantitative PCR to evaluate changes in gene expression of these enzymes in HT29 and VK2/E6E7 mucosal epithelial cells after treatment with Lactobacillus and uropathogenic bacteria. We have found that bacterial treatment downregulates gene expression of elements responsible for miRNA biogenesis, and our results showed different responses dependent on the cell line. In addition to this we have also determined stable reference genes for use in further studies involving this model. Our findings indicate that modulation of the RNAi machinery might be an important element of immune regulation by bacterial colonists.
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Kalia, Munishikha [Verfasser]. "Insights into the functioning of Argonaute proteins with the aid of molecular dynamics simulations / Munishikha Kalia." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2014. http://d-nb.info/106406387X/34.
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Hauptmann, Judith [Verfasser], and Gunter [Akademischer Betreuer] Meister. "Human Argonaute proteins: Analysis of endonucleolytic activity and endogenous phosphorylation sites / Judith Hauptmann ; Betreuer: Gunter Meister." Regensburg : Universitätsbibliothek Regensburg, 2015. http://d-nb.info/1122354584/34.
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Comazzetto, Stefano. "Functional analysis of the Argonaute family proteins during early embryonic and germ cell development in the mouse." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/2c66535f-6b71-4065-8bd5-a3b513711a76.
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Argonaute proteins are known regulators of gene expression through their association with small non-coding RNAs. Specifically in mammals, microRNAs (miRNA) and short interfering RNAs (siRNAs) bind to Ago-like subclade of the Argonaute family to regulate gene expression, while Piwi-interacting RNAs (piRNAs) associate with PIWI proteins to silence transposons in the germ line. In mammals, genetic experiments revealed that Ago2 is the central effector for both siRNA and miRNA silencing pathways in several developmental processes. Phosphorylation of serine 388 of murine protein is dependent on the MK2 and Akt/PKB pathways and regulates Ago2 localization. I created a nonphoshorylatable Ago2S388A allele to study this phosphorylation event during mouse development. Lack of Ago2 phosphorylation is dispensable for adult hematopoiesis, oogenesis, and for the MK2-dependent endotoxic stress response. Furthermore, I discovered a partially penetrant lethal phenotype of Ago2S388A/S388A embryos at peri-implantation in mixed 129P2xC57Bl6N and 129S2 genetic backgrounds, but not in C57Bl/6N background. In conclusion, I demonstrated for the first time that mutation of a post-translational modification site has a differential phenotypic outcome depending on the genetic background. Genetic ablation of PIWI-proteins in the mouse leads to a block in male meiosis. Strikingly, loss of Miwi2 causes an additional progressive loss of germ cells, reminiscent of a stem cell phenotype. However, Miwi2 levels are high in fetal testis and become undetectable soon after birth. I generated a Miwi2tdTomato transcriptional reporter allele and proved that Miwi2 expression marks a subpopulation of spermatogonial cells that possesses surface markers, cell cycle and gene expression profile reminiscent of stem cells in juvenile testis. Comparison of juvenile and adult Miwi2-expressing cells revealed a significant difference in Thy-1 surface expression, which could be indicative of a higher self-renewal capacity in young cells. In summary, these data posit that Miwi2 expression defines a population of bona fide stem cells in the mouse testis.
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Innarella, Maria Rosaria [Verfasser]. "Investigation of the effect of human Argonaute proteins on the maturation of short haipin RNAs / Maria Rosaria Innarella." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2015. http://d-nb.info/106640139X/34.
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Wee, Liang Meng. "RNA Interference by the Numbers: Explaining Biology Through Enzymology: A Dissertation." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/661.
Full textAbstract:
Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct?
To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3' supplementary and tail. Of these domains, the seed initiates binding to target.
Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates.
Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets.
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Wee, Liang Meng. "RNA Interference by the Numbers: Explaining Biology Through Enzymology: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/661.
Full textAbstract:
Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct?
To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3' supplementary and tail. Of these domains, the seed initiates binding to target.
Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates.
Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets.
17
Zander, Adrian [Verfasser], and Dina [Akademischer Betreuer] Grohmann. "Biochemische Charakterisierung des archäellen Argonaute-Proteins aus Methanocaldococcus jannaschii und strukturelle Untersuchungen mit Einzelmolekül-Förster-Resonanz-Energie-Transfer-Studien / Adrian Zander ; Betreuer: Dina Grohmann." Regensburg : Universitätsbibliothek Regensburg, 2018. http://d-nb.info/1153606550/34.
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Wang, Wei. "Unveiling Molecular Mechanisms of piRNA Pathway from Small Signals in Big Data: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/805.
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PIWI-interacting RNAs (piRNA) are a group of 23–35 nucleotide (nt) short RNAs that protect animal gonads from transposon activities. In Drosophila germ line, piRNAs can be categorized into two different categories— primary and secondary piRNAs— based on their origins. Primary piRNAs, generated from transcripts of specific genomic regions called piRNA clusters, which are enriched in transposon fragments that are unlikely to retain transposition activity. The transcription and maturation of primary piRNAs from those cluster transcripts are poorly understood. After being produced, a group of primary piRNAs associates Piwi proteins and directs them to repress transposons at the transcriptional level in the nucleus. Other than their direct role in repressing transposons, primary piRNAs can also initiate the production of secondary piRNA. piRNAs with such function are loaded in a second PIWI protein named Aubergine (Aub). Similar to Piwi, Aub is guided by piRNAs to identify its targets through base-pairing. Differently, Aub functions in the cytoplasm by cleaving transposon mRNAs. The 5' cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3' ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5' ends are generated by another PIWI protein are named secondary piRNAs. Intriguingly, secondary piRNAs loaded into Ago3 also cleave transposon mRNA or piRNA cluster transcripts and produce more secondary piRNAs loaded into Aub. This reciprocal feed-forward loop, named the “Ping-Pong cycle”, amplified piRNA abundance.
By dissecting and analyzing data from large-scale deep sequencing of piRNAs and transposon transcripts, my dissertation research elucidates the biogenesis of germline piRNAs in Drosophila.
How primary piRNAs are processed into mature piRNAs remains enigmatic. I discover that primary piRNA signal on the genome display a fixed periodicity of ~26 nt. Such phasing depends on Zucchini, Armitage and some other primary piRNA pathway components. Further analysis suggests that secondary piRNAs bound to Ago3 can initiate phased primary piRNA production from cleaved transposon RNAs. The first ~26 nt becomes a secondary piRNA that bind Aub while the subsequent piRNAs bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. This discovery adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. We further find that most Piwi-associated piRNAs are generated from the cleavage products of Ago3, instead of being processed from piRNA cluster transcripts as the previous model suggests. The cardinal function of Ago3 is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather to make piRNAs that guide post-transcriptional silencing by Aub. Although Ago3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. The alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.
The Ping-Pong model depicts that first ten nucleotides of Aub-bound piRNAs are complementary to the first ten nt of Ago3-bound piRNAs. Supporting this view, piRNAs bound to Aub typically begin with Uridine (1U), while piRNAs bound to Ago3 often have adenine at position 10 (10A). Furthermore, the majority of Ping-Pong piRNAs form this 1U:10A pair. The Ping-Pong model proposes that the 10A is a consequence of 1U. By statistically quantifying those target piRNAs not paired to g1U, we discover that 10A is not directly caused by 1U. Instead, fly Aub as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs. On the other hand, this t1A (and g10A after loading) piRNA directly give rise to 1U piRNA in the next Ping-Pong cycle, maximizing the affinity between piRNAs and PIWI proteins.
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Wang, Wei. "Unveiling Molecular Mechanisms of piRNA Pathway from Small Signals in Big Data: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/805.
Full textAbstract:
PIWI-interacting RNAs (piRNA) are a group of 23–35 nucleotide (nt) short RNAs that protect animal gonads from transposon activities. In Drosophila germ line, piRNAs can be categorized into two different categories— primary and secondary piRNAs— based on their origins. Primary piRNAs, generated from transcripts of specific genomic regions called piRNA clusters, which are enriched in transposon fragments that are unlikely to retain transposition activity. The transcription and maturation of primary piRNAs from those cluster transcripts are poorly understood. After being produced, a group of primary piRNAs associates Piwi proteins and directs them to repress transposons at the transcriptional level in the nucleus. Other than their direct role in repressing transposons, primary piRNAs can also initiate the production of secondary piRNA. piRNAs with such function are loaded in a second PIWI protein named Aubergine (Aub). Similar to Piwi, Aub is guided by piRNAs to identify its targets through base-pairing. Differently, Aub functions in the cytoplasm by cleaving transposon mRNAs. The 5' cleavage products are not degraded but loaded into the third PIWI protein Argonaute3 (Ago3). It is believed that an unidentified nuclease trims the 3' ends of those cleavage products to 23–29 nt, becoming mature piRNAs remained in Ago3. Such piRNAs whose 5' ends are generated by another PIWI protein are named secondary piRNAs. Intriguingly, secondary piRNAs loaded into Ago3 also cleave transposon mRNA or piRNA cluster transcripts and produce more secondary piRNAs loaded into Aub. This reciprocal feed-forward loop, named the “Ping-Pong cycle”, amplified piRNA abundance.
By dissecting and analyzing data from large-scale deep sequencing of piRNAs and transposon transcripts, my dissertation research elucidates the biogenesis of germline piRNAs in Drosophila.
How primary piRNAs are processed into mature piRNAs remains enigmatic. I discover that primary piRNA signal on the genome display a fixed periodicity of ~26 nt. Such phasing depends on Zucchini, Armitage and some other primary piRNA pathway components. Further analysis suggests that secondary piRNAs bound to Ago3 can initiate phased primary piRNA production from cleaved transposon RNAs. The first ~26 nt becomes a secondary piRNA that bind Aub while the subsequent piRNAs bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. This discovery adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. We further find that most Piwi-associated piRNAs are generated from the cleavage products of Ago3, instead of being processed from piRNA cluster transcripts as the previous model suggests. The cardinal function of Ago3 is to produce antisense piRNAs that direct transcriptional silencing by Piwi, rather to make piRNAs that guide post-transcriptional silencing by Aub. Although Ago3 slicing is required to efficiently trigger phased piRNA production, an alternative, slicing-independent pathway suffices to generate Piwi-bound piRNAs that repress transcription of a subset of transposon families. The alternative pathway may help flies silence newly acquired transposons for which they lack extensively complementary piRNAs.
The Ping-Pong model depicts that first ten nucleotides of Aub-bound piRNAs are complementary to the first ten nt of Ago3-bound piRNAs. Supporting this view, piRNAs bound to Aub typically begin with Uridine (1U), while piRNAs bound to Ago3 often have adenine at position 10 (10A). Furthermore, the majority of Ping-Pong piRNAs form this 1U:10A pair. The Ping-Pong model proposes that the 10A is a consequence of 1U. By statistically quantifying those target piRNAs not paired to g1U, we discover that 10A is not directly caused by 1U. Instead, fly Aub as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs. On the other hand, this t1A (and g10A after loading) piRNA directly give rise to 1U piRNA in the next Ping-Pong cycle, maximizing the affinity between piRNAs and PIWI proteins.
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Mauri, Marta. "Interactome of TNRC6 W-motifs and their conserved Role in miRNA-mediated silencing." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18616.
Full textAbstract:
MicroRNAs (miRNAs) sind kurze nicht-kodierende RNAs, die auf posttranskriptionaler Ebene die Genexpression hemmen. Dafür bilden miRNAs Ribonukleoprotein-Komplexe, deren Kernbestandteile aller Bilateria Argonaute (AGO) und GW182 /TNRC6 Proteine sind. GW182 / TNRC6-Proteine rekrutieren CCR4-NOT-Deadenylasen über kurze Tryptophan-reiche Motive (W-Motive), welche additiv wirken und fördern so die translationale Repression und den Abbau von Ziel-mRNAs. Um mehr über die Mechanismen der miRNA-abhängigen Genrepression zu erfahren, habe ich W-Motiv-abhängige Interaktionspartner humaner TNRC6C Proteine bestimmt. Hierzu habe ich, mithilfe von quantitativer Massenspektrometrie, das Interaktom von wildtyp TNRC6C Proteinen mit dem von TNRC6C Proteinen, deren W-Motive mutiert wurden, verglichen. Neben bekannten Interaktionspartnern, wie Untereinheiten des CCR4-NOT Komplexes, habe ich Komponenten von Clathrin-Vesikeln (CCVs), Stoffwechsel assoziierte Enzyme, mitochondriale Proteine, RNA Helikasen, Kinasen und Phosphatasen mit potentiellen Funktionen in der miRNA-assoziierten Repression identifiziert. Die im ersten Teil dieser Studie vorgestellten Ergebnisse legen nahe, dass CCVs die Speicherung oder das Recycling von TNRC6 und AGO Proteinen vermitteln können und somit das miRNA-Silencing modulieren.
Der zweite Teil dieser Studie befasst sich mit der Konservierung von miRNA vermitteltem Gen-Silencing in Cnidaria (Nematostella vectensis), welche sich vor 600 Millionen Jahren von der Ahnenreihe der Metazoa abspalteten. Hier zeige ich anhand humaner Zellen, dass Nematostella GW182, ähnlich wie in Bilateria, von AGO rekrutiert wird und nachfolgend in der Repression der mRNA fungiert, was darauf hinweist, dass dieser Mechanismus der miRNA-vermittelten Geninhibition bereits in den letzten gemeinsamen Vorfahren von Cnidaria und Bilateria aktiv war.MicroRNAs (miRNAs) are short non-coding RNAs that act as post-transcriptional repressors of gene expression. To function miRNAs are assembled in ribonucleoprotein complexes, whose core components in bilaterian animals are Argonaute (AGO) and GW182/TNRC6 proteins. GW182/TNRC6 proteins additively recruit CCR4-NOT deadenylases via short tryptophan-containing motifs (W-motifs), thereby promoting translational repression and the decay of target mRNAs. To gain deeper insights into the mechanisms of miRNA silencing I determined the W-motif-specific interactome of human TNRC6C proteins. Using Stable Isotope Labeling by Amino acids in Cell Culture (SILAC) coupled to affinity purification and Mass Spectrometry (MS) I identified proteins enriched with wild type TNRC6C as compared to two mutants with disrupted W-motifs. Besides known functional interactors, such as subunits of the CCR4-NOT complex, I identified several components of clathrin-coated vesicles (CCVs), metabolic enzymes, mitochondrial proteins, RNA helicases, kinases, and phosphatases with potential functional roles in miRNA-mediated repression. The results presented in the first part of this thesis indicate that CCVs may mediate the storage or recycling of TNRC6 and AGO proteins, thus modulating miRNA silencing. The second part of the thesis addressed the conservation of the mechanisms of miRNA silencing via W-motifs in the cnidarian Nematostella vectensis, separated by 600 million years from other Metazoa. Using cultured human cells, I showed that similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with AGO proteins, and functions downstream to repress mRNA, indicating that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.
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Minoia, Sofia. "Degradación in vivo de un viroide de replicación nuclear: rutas catalizadas por proteínas Argonauta cargadas con pequeños RNAs viroidales y por otras ribonucleasas que generan RNAs subgenómicos." Doctoral thesis, Universitat Politècnica de València, 2015. http://hdl.handle.net/10251/48553.
Full textAbstract:
Los viroides, los agentes infecciosos más simples de la escala biológica, están constituidos por
una molécula circular de RNA monocatenario de aproximadamente 250-400 nucleótios (nt) que no
codifica proteína alguna. A pesar de esta simplicidad estructural, los viroides son capaces de
replicarse autónomamente, moverse sistémicamente y en muchos casos causar enfermedades en
sus plantas huéspedes. Las infecciones producidas por viroides representativos generan la
acumulación de pequeños RNAs viroidales (vd-sRNAs) de 21-24 nt con características similares a
los pequeños RNA interferentes (siRNAs), la huella dactilar del silenciamiento mediado por RNA.
La identificación de los vd-sRNAs implica que los viroides son diana de la primera barrera de
silenciamiento mediado por RNA, formada por las RNasas ‘Dicer-like’ (DCLs). Para examinar si
los vd-sRNAs se unen a las proteínas AGOs —el componente clave del complejo RISC (‘RNAinduced
silencing complex’) que constituye la segunda barrera del silenciamiento mediado por
RNA— hojas de Nicotiana benthamiana infectadas por el viroide del tubérculo fusiforme de la
patata (PSTVd) se agroinfiltraron con nueve de las diez proteínas AGOs de Arabidopsis thaliana.
Inmunoprecipitaciones a partir de los halos agroinfiltrados y análisis ‘Western-’ y ‘Northern-blot’
han mostrado que todas las AGOs se expresaron y, a excepción de AGO6, AGO7 y AGO10,
unieron vd-sRNAs: AGO1, AGO2 y AGO3 los de 21 y 22 nt, mientras que AGO4, AGO5 y AGO9
también mostraron afinidad por los de 24 nt. La secuenciación masiva mostró que las AGO1,
AGO2, AGO4 y AGO5 agroexpresadas unen los PSTVd-sRNAs en función de su tamaño y
nucleótido 5’-terminal, y que los perfiles de los correspondientes vd-sRNAs cargados en las AGOs
adoptan una distribución específica a lo largo del genoma viroidal. La agroexpresión de AGO1,
AGO2, AGO4 y AGO5 en hojas de N. benthamiana infectadas con PSTVd atenuó la acumulación
de los RNAs genómico viroidales, indicando que éstos, o sus precursores, también son diana de
RISC. En contraste con los ribovirus, la infección de PSTVd en N. benthamiana no afectó de forma
significativa la regulación mediada por miR168 de la AGO1 endógena, que carga vd-sRNAs con
especificidad similar a su homóloga de A. thaliana.
Mientras se conoce bien la biogénesis de los RNA viroidales, su degradación está restringida a
algunos datos que implican al silenciamiento mediado por RNA. En el curso de nuestros estudios
sobre el PSTVd, hemos observado consistentemente un patrón de 6-7 RNAs subgénomicos
(sgRNAs) de polaridad (+) que aparecen junto con los RNAs monoméricos circulares y lineares en
berenjena, un huésped experimental de este viroide. Hibridaciones ‘Northern-blot’ con sondas de
tamaño parcial y completo, mostraron que los sgRNAs (+) de PSTVd derivan de diferentes
regiones del RNA genómico y que algunos son parcialmente solapantes. Parte de los sgRNAs (+)
de PSTVd se observaron también en N. benthamiana y tomate, donde han pasado desapercibidos a
causa de su menor acumulación. El análisis por extensión de cebador de sgRNAs (+) de PSTVd
representativos excluye que sean productos de terminaciones prematuras de la transcripción, pues
carecen del extremo 5’ común que cabría esperar si ésta empezara en una posición específica.
Ulteriores análisis mediante 5’- y 3’-RACE indican que los sgRNAs (+) de PSTVd tienen extremos
5’-OH y 3’-P, que probablemente resultan de cortes endonucleolíticos de precursores más largos
catalizados por RNasas típicas que generan este tipo de extremos. Análisis de sgRNA (-) de
PSTVd, que también se acumulan en berenjena infectada, mostraron que presentan características
estructurales muy similares a los sgRNA (+). Nuestros resultados proporcionan una nueva visión
de cómo ocurre la degradación in vivo de los RNAs viroidales, posiblemente durante su replicación,
y sugieren que síntesis y degradación de las cadenas de PSTVd están conectadas, como se ha
observado en los mRNAs.Minoia, S. (2015). Degradación in vivo de un viroide de replicación nuclear: rutas catalizadas por proteínas Argonauta cargadas con pequeños RNAs viroidales y por otras ribonucleasas que generan RNAs subgenómicos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48553
TESIS
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Conine, Colin C. "Small RNAs and Argonautes Provide a Paternal Epigenetic Memory of Germline Gene Expression to Promote Thermotolerant Male Fertility: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/724.
Full textAbstract:
During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.
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Conine, Colin C. "Small RNAs and Argonautes Provide a Paternal Epigenetic Memory of Germline Gene Expression to Promote Thermotolerant Male Fertility: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/724.
Full textAbstract:
During each life cycle, gametes must preserve and pass on both genetic and epigenetic information, making the germline both immortal and totipotent. In the male germline the dramatic morphological transformation of a germ cell through meiosis, into a sperm competent for fertilization, while retaining this information is an incredible example of cellular differentiation. This process of spermatogenesis is inherently thermosensitive in numerous metazoa ranging from worms to man. Here, I describe the role of two redundant AGO-class paralogs, ALG-3/4, and their small RNA cofactors, in promoting thermotolerant male fertility in Caenorhabditis elegans. alg-3/4 double mutants exhibit temperature dependent sterility resulting from defective spermiogenesis, the postmeiotic differentiation of haploid spermatids into spermatozoa competent for fertilization. The essential Argonaute CSR-1 functions with ALG-3/4 to positively regulate target genes required for spermiogenesis by promoting transcription via a small RNA positive feedback loop. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal loaded into CSR-1 to maintain transcriptionally active chromatin at genes required for spermiogenesis and to provide an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. The ALG-3/4 small RNA pathway also initiates silencing small RNA signals loaded into WAGO vii Argonautes, which function to posttranscripitonally silence their target mRNAs. Silencing WAGO/small RNA-complexes are present in sperm and presumably transmitted to offspring upon fertilization. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic activating and silencing signals in the form of Argonaute/small-RNA complexes, constituting a memory of gene expression in preceding generations.
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Schraivogel, Daniel Verfasser], and Gunter [Akademischer Betreuer] [Meister. "miR-9/9* regulate the tumor suppressor CAMTA1 in glioblastoma stem cells - Part I. Nuclear transport of Argonaute and TNRC6 proteins - Part II / Daniel Schraivogel. Betreuer: Gunter Meister." Regensburg : Universitätsbibliothek Regensburg, 2015. http://d-nb.info/1077648421/34.
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Han, Bo W. "Using Experimental and Computational Strategies to Understand the Biogenesis of microRNAs and piRNAs: A Dissertation." eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/782.
Full textAbstract:
Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies.
The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation.
In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.
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Han, Bo W. "Using Experimental and Computational Strategies to Understand the Biogenesis of microRNAs and piRNAs: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/782.
Full textAbstract:
Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies.
The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation.
In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.
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McCue, Andrea D. "Transposable element RNAi goes beyond post-transcriptional silencing: mRNA-derived small RNAs both regulate genes and initiate DNA methylation." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437481732.
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Jackel, Jamie Nicole. "GEMINIVIRUSES AS MODELS TO STUDY THE ESTABLISHMENT AND MAINTENANCE OF DNA METHYLATION." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1367494030.
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Rüdel, Sabine. "Biochemische Studien zur Phosphorylierung humaner Argonaute-Proteine." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-126560.
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Weinmann, Lasse. "Functional characterisation of microRNA-containing Argonaute protein complexes." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-103249.
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Ender, Christine. "Characterization of protein and small RNA components of Argonaute complexes." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-124199.
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Bamezai, Shiva [Verfasser]. "Characterizing the functional role of argonaute protein PIWIL4 in acute myeloid leukemia / Shiva Bamezai." Ulm : Universität Ulm. Medizinische Fakultät, 2015. http://d-nb.info/1069960780/34.
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Salem, Esam. "The Functional Role of Hepatic Argonaute (Ago)-2 Slicer Activity in Metformin’s Action and Glucose Metabolism in Obese Mice." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595845797361702.
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Deerberg, Andrea [Verfasser]. "Biochemische Charakterisierung der siRNA-vermittelten Erkennung und Spaltung von target RNA durch humanes Argonaute2-Protein / Andrea Deerberg." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2012. http://d-nb.info/1021332771/34.
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Boesler, Benjamin [Verfasser]. "Molekularbiologische Charakterisierung der Argonauten Proteine AgnA und AgnB innerhalb der RNA vermittelten Genregulation in D. discoideum / Benjamin Boesler." Kassel : Universitätsbibliothek Kassel, 2012. http://d-nb.info/1035209810/34.
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Oellig, Christine [Verfasser], Sabine [Akademischer Betreuer] [Gutachter] Schneider, Tobias A. M. [Gutachter] Gulder, and Michael [Gutachter] Groll. "Structural Characterization of the Argonaute Protein from Methanocaldococcus jannaschii / Christine Oellig ; Gutachter: Tobias A. M. Gulder, Michael Groll, Sabine Schneider ; Betreuer: Sabine Schneider." München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1147565457/34.
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Janas, Maja. "Novel Regulation of MicroRNA Biogenesis and Function." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10121.
Full textAbstract:
MicroRNAs are small noncoding RNAs that post-transcriptionally reduce protein output from most human mRNAs by mechanisms that are still obscure. This thesis provides insights into three aspects of microRNA biogenesis and function described below. MicroRNA precursors are excised from primary transcripts by the Microprocessor complex containing Drosha and DGCR8. Although most microRNAs are located in introns of protein-coding and noncoding genes, the mechanisms coordinating microprocessing and splicing are unclear. MiR-211 is a microRNA expressed from intron 6 of melastatin, a suspected melanoma tumor suppressor. We demonstrate that miR-211, and not melastatin, is responsible for the tumor suppressive function of this locus, that Drosha-mediated processing of the miR-211 precursor promotes splicing of melastatin exon 6-exon 7 junctions, and that perturbing 5' splice site recognition by the U1 snRNP reduces Drosha recruitment to intron 6 specifically and intronic microRNA levels globally. Thus we identify a novel physical and functional coupling between microprocessing and splicing. Typically, Agos stabilize mature microRNAs and as a complex stoichiometrically bind to complementary mRNAs. We demonstrate an alternative order of events in which Agos bind and repress pre-formed imperfect microRNA-mRNA duplexes in processing bodies of live cells, and cleave pre-formed perfect microRNA-mRNA duplexes in vitro. Our data support a novel catalytic model whereby Agos first deposit microRNAs onto mRNAs and dissociate, thus priming multiple microRNA-mRNA duplexes for concurrent repression by a single Ago. Despite key roles in development and pathogenesis, effectors and regulators of microRNA-mediated repression are still poorly characterized. An RNAi screen revealed that depletion of ribosomal proteins of either small or large ribosomal subunit dissociates microRNA-containing complexes from mRNAs repressed at translation initiation, increasing their polysome association, translation, and stability relative to untargeted mRNAs. Thus ribosomal proteins globally regulate microRNA function. Another RNAi screen revealed that Akt3 phosphorylates Ago2, which negatively regulates cleavage and positively regulates translational repression of microRNA-targeted mRNAs. Thus Ago2 phosphorylation is a molecular switch between its mRNA cleavage and translational repression activities. The following pages will place these novel insights into biological and disease-relevant context, will describe what was known prior to these studies, and will provide perspectives for future studies.
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Völler, Daniel Reinhold [Verfasser], and Anja-Katrin [Akademischer Betreuer] Bosserhoff. "Die Rolle der Argonaut Proteine in der Entstehung und Progression des malignen Melanoms / Daniel Reinhold Völler. Betreuer: Anja-Katrin Bosserhoff." Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1083248243/34.
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Daschkey, Svenja Verfasser], Arndt [Akademischer Betreuer] [Borkhardt, and Martin [Akademischer Betreuer] Lercher. "microRNA expression profiling of pediatric acute myeloid leukemia patient samples and global identification of Argonaute protein-associated RNAs in respective cell line models / Svenja Daschkey. Gutachter: Martin Lercher. Betreuer: Arndt Borkhardt." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2012. http://d-nb.info/1021781347/34.
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Mengardi, Chloé. "Étude de l'effet des microARN sur l'initiation de la traduction dirigée par l'IRES du Virus de l'Hépatite C." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEN001.
Full textAbstract:
Les microARN (miARN) sont de petits ARN non-codants qui contrôlent l’expression génique, en s’hybridant, le plus souvent, de manière imparfaite à des séquences spécifiques qui se trouvent généralement dans la région non traduite en 3' (3’UTR) de transcrits cibles. Les miARN guident sur l’ARN messager (ARNm) un complexe protéique appelé RNA-induced Silencing Complex (RISC), composé des protéines Argonaute et TNRC6, qui perturbe l’initiation de la traduction et provoque la déadénylation et la dégradation du transcrit. C'est l’interaction entre le RISC et le complexe de pré-initiation de la traduction 43S (composé de la petite sous-unité ribosomique 40S et des facteurs d’initiation associés) qui entraîne la répression traductionnelle de l’ARNm ciblé. Des résultats récents ont démontré que le RISC perturbe le balayage de la région non traduite en 5’ (5’UTR) par le ribosome, étape qui requiert la présence de 2 facteurs d’initiation qui sont eIF4F qui reconnaît et lie la coiffe ainsi que la protéine PABP, fixée le long de la queue poly(A). Toutefois, les miARN peuvent également induire la stimulation de la traduction des transcrits cibles dans les cellules quiescentes, dans un lysat d’embryons de drosophiles ou encore dans les ovocytes de Xénope. Le mécanisme moléculaire de stimulation de l’expression par les miARN est encore mal connu mais requiert l’absence de queue poly(A) en 3’ des ARN cible et de TNRC6 au sein du complexe RISC. Le Virus de l’Hépatite C (VHC) possède en 5’ de son ARNg un site d’entrée interne du ribosome (IRES) qui recrute la petite sous-unité ribosomique 40S, sans nécessiter la reconnaissance de la coiffe par eIF4F, ni la protéine PABP, ni le balayage de la 5’UTR par le ribosome. Ces caractéristiques singulières nous ont conduits à rechercher l'impact du complexe RISC fixé en 3’ de l’ARNm sur l’initiation de la traduction du VHC. Pour cela, nous avons utilisé des transcrits contenant l'IRES du VHC en 5' et des sites d’hybridation du miARN let-7 en 3’. Ces ARNm ont ensuite été transfectés dans des lignées cellulaires hépatocytaires, ou non. A notre grande surprise, nous avons observé que la fixation du miARN let-7 sur la région 3' du transcrit stimulait fortement l’expression dirigée par l’IRES de VHC. Toutefois, l’augmentation de l’expression n’est pas due à la stabilisation du transcrit mais bien à une hausse significative de la synthèse protéique indépendamment d’un quelconque effet de miR-122. En utilisant d’autres IRES dites 'HCV-like', nous avons pu confirmer ces résultats et démontrer que, l’ajout d’une queue poly(A) en 3’ du transcrit, capable de fixer la PABP, annule cet effet stimulateur suggérant que l’absence de cette protéine est nécessaire pour que le complexe RISC stimule la traduction du VHCMicroRNAs (miRNAs) are small non coding RNAs which control gene expression by recognizing and hybridizing to a specific sequence generally located in the 3’UTR of targeted messenger RNA (mRNA). miRNAs serve as a guide for the RNA-Induced Silencing Complex (RISC) that is composed by, at least, the Argonaute proteins and TNRC6. Recent studies have suggested that translation inhibition occurs first and is then followed by deadenylation and degradation of the targeted transcript. The miRNA-induced inhibition of protein synthesis occurs at the level of translation initiation during the ribosomal scanning step and it requires the presence of both the initiation factor eIF4G and the poly(A) Binding Protein (PABP). In this process, the RISC interacts with both PABP and 43S pre-initiation complex (composed by initiation factors and ribosome) and it results in the disruption of linear scanning of the ribosome along the 5’ Untranslated Region (5’UTR). In some specific cases, the binding of miRNAs to their target sequences can upregulate translation initiation. This has notably been demonstrated in G0 quiescent cells, drosophila embryos and Xenopus oocytes. Although the molecular mechanism by which upregulation occurs remains to be precisely determined, it appears that the absence of a poly(A) tail and the lack of availability of the TNRC6 proteins are amongst the major determinants. In the particular case of the Hepatitis C Virus (HCV), the genomic RNA is uncapped and non polyadenylated and harbors an Internal Ribosome Entry Site (IRES) which directly binds to the ribosome with no need for cap-recognition, PABP binding and ribosome scanning. These peculiar features of the HCV IRES prompted us to investigate how viral translation can be regulated by the miRNA machinery. In order to do that, we have used a mRNA that contains the HCV IRES in 5’ and 4 let-7 binding sites in its 3’ extremity. To most of our surprise, we have observed a strong stimulation of the expression of the HCV IRES when the construct is bearing the let-7 sites. This effect is not due to any interference with the miR-122 binding sites although the magnitude of stimulation reached the same level. Our data show that it is the presence of the RISC on the 3' end of the transcript that can stimulate internal ribosome entry at the 5' end. By using other HCV-like IRESes, we could confirm these data and further showed that the absence of a poly(A) tail was an absolute requirement for the stimulation to occur. These effects are not due to an increase of mRNA stability and are rather exerted at the level of translation
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Hacquard, Thibaut. "Molecular characterization of the F-box protein FBW2 in the RNA silencing in Arabidopsis thaliana." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ069.
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L'ARN interférence est un mécanisme moléculaire conservé chez les Eucaryotes dont les principaux acteurs sont les protéines ARGONAUTE (AGO). Chez les plantes, AGO1 est une protéine essentielle à la croissance et la défense antivirale. Elle utilise des petits ARNs comme sondes pour reconnaître et réguler des ARN messagers. Les virus ont développé des suppresseurs de l'ARN interférence pour surmonter cette défense. L'un d'entre eux, P0 du virus de la mosaïque jaune du navet, est comme une protéine F-box qui détourne le complexe SCF, une ubiquitine ligase E3, et conduit AGO1 vers la protéolyse ubiquitine-dépendante. Cette dégradation utilise la vacuole au lieu du protéasome 26S, généralement associé à la dégradation ubiquitine-dépendante. Ce mécanisme de protéolyse n'est pas compris et est aussi apparent quand AGO1 est déstabilisé de manière endogène, suggérant que P0 utilise une voie déjà existante. Une protéine F-box d'Arabidopsis, FBW2, a été décrite comme impactant l'homéostasie d'AGO1 indépendamment du protéasome. Mon projet de thèse visait à caractériser l'activité F-box de FBW2 et à comprendre la relation entre AGO1 et FBW2 ainsi que ses conséquences sur l'ARN interférence. Les résultats obtenus dans ce manuscrit montrent que le complexe SCFFBW2 interagit avec AGO1 et déclenche sa dégradation via un processus indépendant de l'autophagie ou du protéasome, tout en n'affectant que faiblement l'ARN interférence. FBW2 ciblerait en fait un sous-ensemble de protéines AGO1 qui semble ne pas contenir de petits ARNs. Cette régulation jouerait un rôle de surveillance pour prévenir une activité délétère d'AGO1 en absence de petits ARNsRNA silencing is a conserved molecular mechanism in eukaryotes, of which the main effectors are the ARGONAUTE (AGO) proteins. In plants, AGO1 is a protein that is essential for growth and antiviral defence. It uses small RNAs as probe to recognize and regulate messenger RNAs. Viruses have developed suppressors of RNA silencing to overcome this defence. One of these, P0 from the Turnip Yellows Virus, acts as an F-box protein to hijack the SCF complex, an E3 ubiquitin ligase, and guide AGO1 to the ubiquitin-dependent proteolysis. This degradation uses the vacuole instead of the 26S proteasome, generally associated with ubiquitin-dependant proteolysis. This proteolysis mechanism is not understood and is also apparent when AGO1 is endogenously destabilized, suggesting that P0 uses an already existing pathway. An Arabidopsis F-box protein, FBW2, has been shown to impact AGO1 homeostasis independently from the proteasome. My PhD project aimed at characterizing FBW2 F-box activity and understanding the relationship between AGO1 and FBW2, as well as its consequences on the RNA silencing. The results obtained in this manuscript show that the SCFFBW2 interacts with AGO1 and triggers its degradation through an autophagy- and proteasome- independent process, while only weakly affecting the RNA silencing. FBW2 would actually target a subset of AGO1 proteins, which appears not to contain small RNAs. This regulation would play a surveillance role in order to prevent a deleterious activity of AGO1 in absence of small RNAs
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Kuo, Hsun-Chuan, and 郭巡銓. "Putative interacting proteins of ARGONAUTE in Arabidopsis." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/ym98q3.
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碩士國立臺灣大學
微生物與生化學研究所
98
Abstract RNA silencing is important in the growth and development of plants. The major components of RNA silencing complex are different small RNA and ARGONAUTE (AGO) family. Ten AGOs are present in Arabidopsis. It is expected that different function, degrading the target transcript, inhibiting translation, methylating DNA or histone and defending against virus and insects, are achieved by AGOs in complex with different interacting proteins. In this study, putative interacting proteins of AGO1 or AGO4 were searched using the overexpression mutants, yeast two hybrid assay or co-immunoprecipitatin assay. Due to the presence of two extra bases in the vector pN-TAPa, two knock-down mutants, ago1 and ago4 were obtained instead of the expected overexpression. By yeast two hybrid, two CHR proteins, ATP-dependent chromatin remodeling enzymes, were found to interact with AGO1. Recombinant GST-AGO1 expressed in E.coli was used as bait to find interacting protein from crude extract of Arabidopsis using co-immunoprecipitation. The candidates found in this Co-IP experiment were analyzed by LC-MS/MS. They are clathrin binding protein (epsin), F-box/FBD/LRR-repeat protein, short-chain dehydragenase/reductase, Ferredoxin-dependent glutamate synthase 1, Glutamine synthetase, ATP synthase subunit alpha and Elongation factor 1-alpha. Further experiments are necessary to further investigate these putative interacting proteins.
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Li, Joye, and 李俊緯. "Analysis of miRNA sorting signals in different human Argonaute proteins." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/8d6ads.
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Rüdel, Sabine [Verfasser]. "Biochemische Studien zur Phosphorylierung humaner Argonaute-Proteine / Sabine Rüdel." 2010. http://d-nb.info/1010299182/34.
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Jankele, Radek. "Analýza krátkých izoforem proteinů Argonaut z myších oocytů." Master's thesis, 2015. http://www.nusl.cz/ntk/nusl-271728.
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AnalysisofshortArgonauteisoformsfrommouseoocytes Abstract: Argonaute proteins carrying small RNAs form the conserved core of RNA silencing mechanisms, which repress viruses, mobile genetic elements, and genes in a sequence specific manner. The microRNA (miRNA) pathway is a dominant mammalian RNA silencing mechanism in somatic cells, which post-transcriptionally regulates large fraction of genes and thereby adjusts protein levels. miRNA-guided Argonautes inhibit translation and induce deadenylation of complementary mRNAs, ultimately resulting in their decay. In contrast to RNA interference (RNAi), which employs Argonaute slicer activity to directly cleave perfectly complementary RNAs, an effective miRNA-mediated mRNA repression requires multiple Argonaute-associated protein factors and enzymes. The miRNA pathway has been implicated in many complex biological processes ranging from organogenesis, stress-response to haematopoiesis or cancer. Surprisingly, canonical miRNAs are not essential for oocytes and early embryonic development in mice. Even the most abundant miRNAs present in mouse oocytes are unable to effectively repress target genes. However, RNAi, which shares key enzymes with the miRNA pathway, is highly active in oocytes and early embryos. The cause of miRNA inactivity in mouse oocytes remains...
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Chang, Chi-Kai, and 張智凱. "Study on the role of Argonaute protein DjAgo2 in planarian regeneration." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/83382098889819592957.
Full textAbstract:
碩士國立臺灣大學
動物學研究所
100
Planarians are capable of regenerating almost every part of their body, including the brain. Regeneration in planarians is mediated by a group of pluripotent adult somatic stem cells, called neoblasts, which can proliferate and differentiate to replace all tissues. These cells are sensitive to γ-irradiation. DjAgo2 of Dugesia japonica is a homolog of hsAgo2, the key factor that regulates gene expression in miRNA and siRNA pathways. In the study, DjAgo2 was cloned, and YFP-tagged DjAgo2 was expressed in HeLa cells. DjAgo2 accumulates at specific foci that contain the P-body component, RCK. This pattern is similar to what we observed with hsAgo2. By whole-mount in situ hybridization (WISH), Djago2 was found to be highly expressed in regions of the brain and the middle dorsal line. Djago2 expression was increased in the blastema and post-blastema during regeneration. RT-qPCR results confirmed that respective Djago2 mRNA expression levels in the blastema and post-blastema were 5 and 2-times higher than that in the control at 3 days post-amputation. WISH data showed that a portion of Djago2-positive cells were depleted by γ-irradiation. We next examined Djago2 expression levels in neoblasts isolated by FACS. Our RT-qPCR data showed that the Djago2 level was higher in neoblasts than that in differentiated cells. Finally, Djago2 was silenced by feeding planarians Djago2 dsRNA. Depletion of Djago2 resulted in loss of the regeneration capacity and defects in tissue homeostasis in planarians. Djago2 silencing reduced levels of DjpiwiA, Djpcna, and DjpiwiC. Our data also showed that the ratio of M-phase cells was decreased and the ratio of G2-phase cells was increased in Djago2-silenced animals. This suggests that Djago2 silencing blocked the mitosis of neoblasts. Differentiation restriction was also observed in Djago2-silenced animals. Our data indicate that Djago2 is required for neoblast proliferation, and differentiation.
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Ender, Christine [Verfasser]. "Characterization of protein and small RNA components of Argonaute complexes / Christine Ender." 2009. http://d-nb.info/1009587870/34.
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Weinmann, Lasse [Verfasser]. "Functional characterisation of microRNA-containing argonaute protein complexes / vorgelegt von Lasse Weinmann." 2009. http://d-nb.info/995738882/34.
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Wasserman, Gregory Alexander. "A discrete population of ciliated cells express the piRNA binding protein MIWI2 to regulate lung inflammation." Thesis, 2016. https://hdl.handle.net/2144/16735.
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Control of retrotransposon expression in the mammalian germline is regulated by Argonaute family PIWI proteins and their associated small non-coding RNAs known as PIWI-interacting RNAs (piRNAs). To date, no study has demonstrated clear PIWI protein expression nor identified a cellular function(s) for PIWI proteins in the mammalian soma. In contrast to the germline-restricted expression of piRNA associated proteins, we observed that Miwi2 mRNA was induced specifically in epithelial cells during pneumococcal pneumonia. Further investigation showed that similar to its mRNA, MIWI2 protein was indeed expressed outside of the mammalian germline, and was localized to the cytoplasm of a discrete population of multiciliated lung epithelial cells. Immunoprecipitation of MIWI2 from whole lung lysates indicated that it was bound to a small RNA that was longer than a traditional piRNA. Microarray analysis revealed that depletion of MIWI2 in a murine epithelial cell line or in a whole animal model had no effect on retrotransposon expression, further suggesting that lung MIWI2 is independent of nuclear piRNA silencing pathways. Under basal conditions, MIWI2 was required for the normal maintenance of airway epithelial cell fate. In fact, Miwi2 deficiency resulted in an increase in club cells and decrease in ciliated cells indicating that MIWI2 could play a primary role in mucociliary homeostasis or clearance. Similarly, as MIWI2 is induced during lung infection we sought to determine if it participated in host innate immune responses to bacterial infection. Using a clinically relevant model of community acquired pneumonia, Miwi2 deficient mice exhibited an increased expression of inflammatory mediators and immune cell recruitment thus leading to enhanced bacterial clearance. Taken together, these data support the notion that MIWI2 exerts piRNA-independent functions outside of the germline in the ciliated lung epithelium to regulate innate immunity during pneumonia. More broadly, these studies shed light on new areas in PIWI protein and lung ciliated cell biology, and may have implications for multiple diseases including cancer, inflammatory disorders, and infectious diseases.
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Zhang, Xiaoxiao [Verfasser]. "On the function of the Dictyostelium argonaute A protein (AgnA) in epigenetic gene regulation / von Xiaoxiao Zhang." 2006. http://d-nb.info/981053564/34.
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