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https://www.readbyqxmd.com/read/27928938/sequence-and-structure-based-analysis-of-proteins-involved-in-mirna-biogenesis
#1
Chhaya Sharma, Debasisa Mohanty
miRNA biogenesis is a multistage process for generation of a mature miRNA and involves several different proteins. In this work, we have carried out sequence as well as structure based analysis for crucial proteins involved in miRNA biogenesis, namely, Dicer, Drosha, Argonaute (Ago) and Exportin-5 to understand evolution of these proteins in animal kingdom and also to identify key sequence and structural features which are determinants of their function. Our analysis reveals that in animals the miRNA biogenesis pathway first originated in molluscs...
December 8, 2016: Journal of Biomolecular Structure & Dynamics
https://www.readbyqxmd.com/read/27928054/the-influence-of-the-5-terminal-nucleotide-on-agoshrna-activity-and-biogenesis-importance-of-the-polymerase-iii-transcription-initiation-site
#2
Elena Herrera-Carrillo, Zong-Liang Gao, Alex Harwig, Matthias T Heemskerk, Ben Berkhout
Recent evidence indicates that shRNAs with a relatively short basepaired stem do not require Dicer processing, but instead are processed by the Argonaute 2 protein (Ago2). We named these molecules AgoshRNAs as both their processing and silencing function are mediated by Ago2. This alternative processing yields only a single RNA guide strand, which can avoid off-target effects induced by the passenger strand of regular shRNAs. It is important to understand this alternative processing route in mechanistic detail such that one can design improved RNA reagents...
December 7, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27924491/single-molecule-fluorescence-energy-transfer-assays-for-the-characterization-of-reaction-pathways-of-mirna-argonaute-complex
#3
Myung Hyun Jo, Sungchul Hohng
Argonaute proteins are key components of the microRNA-induced silencing complexes (miRISCs) that mediate the posttranscriptional gene silencing of microRNAs and small interfering RNA (siRNAs). The complex reaction mechanism of miRISC is expected to be characterized by tracing the reaction pathways of miRISC at the single-molecule level in real time. In this chapter, we describe single-molecule fluorescence resonance energy transfer (FRET) assays to observe the target binding and reaction pathways of miRISC composed of a recombinant Argonaute and a small RNA...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924490/site-specific-fluorescent-labeling-of-argonaute-for-fret-based-bio-assays
#4
Sarah Willkomm, Adrian Zander, Dina Grohmann
Deciphering the molecular mechanisms of eukaryotic Argonaute proteins is crucial for the understanding of RNA interference (RNAi), a posttranscriptional gene silencing process. Fluorescence-based single-molecule studies like single-molecule Förster resonance energy transfer (FRET) between a donor and acceptor dye represent a versatile tool to gain a mechanistic understanding of the structural dynamics of a biomolecular complex. Until today it was not possible to site-specifically introduce fluorophores into eukaryotic Argonaute...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924489/kinetic-analysis-of-target-rna-binding-and-slicing-by-human-argonaute-2-protein
#5
Sarah Willkomm, Tobias Restle
Analyzing the mechanisms of Argonaute-mediated gene silencing is essential to the understanding of RNA interference (RNAi). RNAi is a process to regulate gene expression on a posttranscriptional level. Directed by single-stranded small RNA guides, Argonaute 2 binds complementary target RNAs, and if the guide displays full complementarity to the targeted sequence, Argonaute 2 slices the bound target RNA. This on the one hand is an important mechanism to regulate gene expression in the cell and on the other hand represents a powerful tool to interfere with harmful gene expression levels...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924488/elucidating-mechanisms-of-molecular-recognition-between-human-argonaute-and-mirna-using-computational-approaches
#6
Hanlun Jiang, Lizhe Zhu, Amélie Héliou, Xin Gao, Julie Bernauer, Xuhui Huang
MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924487/antagonists-of-the-mirna-argonaute-2-protein-complex-anti-mir-agos
#7
Marco F Schmidt, Oliver Korb, Chris Abell
microRNAs (miRNAs) have been identified as high-value drug targets. A widely applied strategy in miRNA inhibition is the use of antisense agents. However, it has been shown that oligonucleotides are poorly cell permeable because of their complex chemical structure and due to their negatively charged backbone. Consequently, the general application of oligonucleotides in therapy is limited. Since miRNAs' functions are executed exclusively by the Argonaute 2 protein, we therefore describe a protocol for the design of a novel miRNA inhibitor class: antagonists of the miRNA-Argonaute 2 protein complex, so-called anti-miR-AGOs, that not only block the crucial binding site of the target miRNA but also bind to the protein's active site...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924486/surface-plasmon-resonance-a-useful-strategy-for-the-identification-of-small-molecule-argonaute-2-protein-binders
#8
Elena Poser, Ilaria Genovese, Silvia Masciarelli, Teresa Bellissimo, Francesco Fazi, Gianni Colotti
Surface plasmon resonance (SPR) is one of the most important techniques for the detection and the characterization of molecular interactions. SPR technology is a label-free approach for monitoring biomolecular interactions in real time. The binding of analytes to molecules immobilized on a thin metal film (ligand) determines a change in the refractive index and, therefore in the angle of extinction of light, is reflected when polarized light hits the film, monitored in real time as a change in the position of the dip in reflected intensity...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924485/small-molecules-targeting-the-mirna-binding-domain-of-argonaute-2-from-computer-aided-molecular-design-to-rna-immunoprecipitation
#9
Teresa Bellissimo, Silvia Masciarelli, Elena Poser, Ilaria Genovese, Alberto Del Rio, Gianni Colotti, Francesco Fazi
The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924484/peptide-based-inhibition-of-mirna-guided-gene-silencing
#10
Johannes Danner, Balagopal Pai, Ludwig Wankerl, Gunter Meister
MicroRNAs (miRNAs) are a large class of small noncoding RNAs that regulate the expression of distinct target mRNAs. miRNAs are incorporated into Argonaute (AGO) proteins and guide them to their target mRNAs. Subsequently, AGO proteins recruit a member of the glycine-tryptophan-rich (GW) protein family by direct protein-protein interaction. GW proteins coordinate all downstream processes leading to robust and efficient gene silencing. A short peptide of GW proteins comprising the AGO interaction motif can be used to biochemically isolate endogenous AGO protein complexes...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924476/competitive-argonaute-based-rna-immunoprecipitation-for-investigation-of-transcriptomic-response-to-anti-mir
#11
John R Androsavich
Identification and validation of microRNA (miRNA) target genes is essential for gaining a better understanding of the many different functions miRNAs have in healthy and diseased cells. From a practical standpoint, validated target genes are also useful for monitoring pharmacological activity of developmental therapeutics that modulate miRNAs, such as anti-miRNA oligonucleotides (anti-miR). Here, we describe a method that uses changes in Argonaute 2-RNA immunoprecipitation in response to competition by anti-miR, titrated ex vivo, as physical evidence for target validation...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924475/determination-of-anti-mir-association-with-mirna-argonaute-complexes-in-vivo
#12
Dimitrios G Zisoulis
Aberrant expression of microRNAs (miRNAs) has been causatively linked to multiple disease pathologies while pharmacological inhibition of overexpressed miRNAs by modified oligonucleotides, termed anti-miRs, has been shown to ameliorate the disease phenotype. Anti-miRs are also widely used in academia to define miRNA-mediated regulation of gene networks in vitro and in vivo. Here, we describe a methodology that allows the determination of the physical association of miRNA inhibitors and their targets in the context of the Argonaute complex in vivo, providing unprecedented insight into the physiological interactions of anti-miRs and the miRNA machinery...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27924474/quantification-of-oligonucleotide-association-with-mirna-argonaute-complexes-in-vitro
#13
Dimitrios G Zisoulis
A major challenge in the development of oligonucleotide-based microRNA (miRNA) inhibitors for therapeutic applications is the identification of candidate designs with strong affinity for the target miRNA in the context of the Argonaute complex. To this effect, distinct chemical modifications are employed along the length of the oligonucleotide aimed at strengthening the interactions with the target miRNA. However, the modification chemistry and placement can inadvertently affect the intrinsic ability of the oligonucleotide to pair with its target in the context of Argonaute...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27918527/induction-and-suppression-of-antiviral-rna-interference-by-influenza-a-virus-in-mammalian-cells
#14
Yang Li, Megha Basavappa, Jinfeng Lu, Shuwei Dong, D Alexander Cronkite, John T Prior, Hans-Christian Reinecker, Paul Hertzog, Yanhong Han, Wan-Xiang Li, Sihem Cheloufi, Fedor V Karginov, Shou-Wei Ding, Kate L Jeffrey
Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens(1). However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago(2), remains unknown(3). Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence(4-7) by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs)(8,9)...
December 5, 2016: Nature Microbiology
https://www.readbyqxmd.com/read/27903911/mirtar2go-a-novel-rule-based-model-learning-method-for-cell-line-specific-microrna-target-prediction-that-integrates-ago2-clip-seq-and-validated-microrna-target-interaction-data
#15
Alireza Ahadi, Gaurav Sablok, Gyorgy Hutvagner
MicroRNAs (miRNAs) are ∼19-22 nucleotides (nt) long regulatory RNAs that regulate gene expression by recognizing and binding to complementary sequences on mRNAs. The key step in revealing the function of a miRNA, is the identification of miRNA target genes. Recent biochemical advances including PAR-CLIP and HITS-CLIP allow for improved miRNA target predictions and are widely used to validate miRNA targets. Here, we present miRTar2GO, which is a model, trained on the common rules of miRNA-target interactions, Argonaute (Ago) CLIP-Seq data and experimentally validated miRNA target interactions...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27903888/sirna-carrying-an-e-vinylphosphonate-moiety-at-the-5-end-of-the-guide-strand-augments-gene-silencing-by-enhanced-binding-to-human-argonaute-2
#16
Elad Elkayam, Rubina Parmar, Christopher R Brown, Jennifer L Willoughby, Christopher S Theile, Muthiah Manoharan, Leemor Joshua-Tor
Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5'- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5'- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5'-(E)-vinylphosphonate (5'-E-VP) guide RNA at 2.5-Å resolution. The structure demonstrates how the 5' binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5'-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27900550/biogenesis-of-diverse-plant-phasirnas-involves-an-mirna-trigger-and-dicer-processing
#17
Reina Komiya
It has been almost 30 years since RNA interference (RNAi) was shown to silence genes via double-stranded RNAs (dsRNAs) in Caenorhabditis elegans (Fire et al. 1998). 20-30-nucleotide (nt) small non-coding RNAs are a key element of the RNAi machinery. Recently, phased small interfering RNAs (phasiRNAs), small RNAs that are generated from a long RNA precursor at intervals of 21 to 26-nt, have been identified in plants and animals. In Drosophila, phasiRNAs are generated by the endonuclease, Zucchini (Zuc), in germlines...
November 29, 2016: Journal of Plant Research
https://www.readbyqxmd.com/read/27899576/the-nuclear-and-organellar-trna-derived-rna-fragment-population-in-arabidopsis-thaliana-is-highly-dynamic
#18
Valérie Cognat, Geoffrey Morelle, Cyrille Megel, Stéphanie Lalande, Jean Molinier, Timothée Vincent, Ian Small, Anne-Marie Duchêne, Laurence Maréchal-Drouard
In the expanding repertoire of small noncoding RNAs (ncRNAs), tRNA-derived RNA fragments (tRFs) have been identified in all domains of life. Their existence in plants has been already proven but no detailed analysis has been performed. Here, short tRFs of 19-26 nucleotides were retrieved from Arabidopsis thaliana small RNA libraries obtained from various tissues, plants submitted to abiotic stress or fractions immunoprecipitated with ARGONAUTE 1 (AGO1). Large differences in the tRF populations of each extract were observed...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27895202/the-lbd12-1-transcription-factor-suppresses-apical-meristem-size-by-repressing-argonaute-10-expression
#19
Weiwei Ma, Fuqing Wu, Peike Sheng, Xiaole Wang, Zhe Zhang, Kunneng Zhou, Huan Zhang, Jinlong Hu, Qibing Lin, Zhijun Cheng, Jiulin Wang, Shanshan Zhu, Xin Zhang, Xiuping Guo, Haiyang Wang, Chuanyin Wu, Jianmin Wan
The shoot apical meristem (SAM) consists of a population of multipotent cells that generate all aerial structures and regenerates itself. SAM maintenance and lateral organ development are regulated by several complex signaling pathways, in which the Argonaute gene mediated pathway plays a key role. One Argonaute gene, AGO10, functions as a miRNA locker that attenuates miR165/166 activity and positively regulates shoot apical meristem development, but little is known about when and how AGO10 is regulated at the transcriptional level...
November 28, 2016: Plant Physiology
https://www.readbyqxmd.com/read/27893851/expression-and-regulation-of-piwil-proteins-and-piwi-interacting-rnas-in-rheumatoid-arthritis
#20
Lenka Pleštilová, Michel Neidhart, Giancarlo Russo, Mojca Frank-Bertoncelj, Caroline Ospelt, Adrian Ciurea, Christoph Kolling, Renate E Gay, Beat A Michel, Jiří Vencovský, Steffen Gay, Astrid Jüngel
OBJECTIVE: The PIWIL (P-element induced wimpy testis like protein) subfamily of argonaute proteins is essential for Piwi-interacting RNA (piRNA) biogenesis and their function to silence transposons during germ-line development. Here we explored their presence and regulation in rheumatoid arthritis (RA). METHODS: The expression of PIWIL genes in RA and osteoarthritis (OA) synovial tissues and synovial fibroblasts (SF) was analysed by Real-time PCR, immunofluorescence and Western blot...
2016: PloS One
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