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https://www.readbyqxmd.com/read/29040713/human-argonaute3-has-slicer-activity
#1
Mi Seul Park, Hong-Duc Phan, Florian Busch, Samantha H Hinckley, James A Brackbill, Vicki H Wysocki, Kotaro Nakanishi
Of the four human Argonaute (AGO) paralogs, only AGO2 has been shown to have slicer activity. The others (AGO1, AGO3 and AGO4) have been thought to assemble with microRNAs to form slicer-independent effector complexes that bind target mRNAs and silence gene expression through translational repression and deadenylation but not cleavage. Here, we report that recombinant AGO3 loaded with miR-20a cleaves complementary target RNAs, whereas AGO3 loaded with let-7a, miR-19b or miR-16 does not, indicating that AGO3 has slicer activity but that this activity depends on the guide RNA...
October 11, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29040304/characterization-of-the-zika-virus-induced-small-rna-response-in-aedes-aegypti-cells
#2
Margus Varjak, Claire L Donald, Timothy J Mottram, Vattipally B Sreenu, Andres Merits, Kevin Maringer, Esther Schnettler, Alain Kohl
RNA interference (RNAi) controls arbovirus infections in mosquitoes. Two different RNAi pathways are involved in antiviral responses: the PIWI-interacting RNA (piRNA) and exogenous short interfering RNA (exo-siRNA) pathways, which are characterized by the production of virus-derived small RNAs of 25-29 and 21 nucleotides, respectively. The exo-siRNA pathway is considered to be the key mosquito antiviral response mechanism. In Aedes aegypti-derived cells, Zika virus (ZIKV)-specific siRNAs were produced and loaded into the exo-siRNA pathway effector protein Argonaute 2 (Ago2); although the knockdown of Ago2 did not enhance virus replication...
October 17, 2017: PLoS Neglected Tropical Diseases
https://www.readbyqxmd.com/read/29031931/global-analysis-of-ago2-bound-rnas-reveals-that-mirnas-induce-cleavage-of-target-rnas-with-limited-complementarity
#3
Eunsun Jung, Youngmo Seong, Bohyun Jeon, Hoseok Song, Young-Soo Kwon
Among the four Argonaute family members in mammals, only AGO2 protein retains endonuclease activity and facilitates cleavage of target RNAs base-pairing with highly complementary guide RNAs. Despite the deeply conserved catalytic activity, only a small number of targets have been reported to extensively base pair with cognate miRNAs to be cleaved by AGO2. Here, we analyzed AGO2-bound RNAs by CrossLinking ImmunoPrecipitation (CLIP) of genetically modified cells that express epitope-tagged AGO2 from the native genomic locus...
October 11, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29030851/cellular-approaches-in-investigating-argonaute2-dependent-rna-silencing
#4
Cai Zhang, Joonbae Seo, Takahisa Nakamura
In mammals, there are four Argonaute (Ago) family proteins that play crucial roles in RNA silencing, a process wherein microRNA (miRNA) mediates inhibition of target mRNA translation. Among the Ago proteins, Argonaute2 (Ago2) uniquely possesses an endoribonuclease (slicer) activity that is critical for the biogenesis of specific miRNAs and mRNA cleavage. This Ago2 slicer activity is required for postnatal development. Despite its important roles, there are still gaps in our understanding of the mechanistic basis of Ago2's unique functions in vivo due to a limited availability of experimental tools...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29030847/single-molecule-analysis-for-risc-assembly-and-target-cleavage
#5
Hiroshi M Sasaki, Hisashi Tadakuma, Yukihide Tomari
RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29028450/comparing-two-approaches-of-mir-34a-target-identification-biotinylated-mirna-pulldown-vs-mirna-overexpression
#6
Hassaan Mehboob Awan, Abdullah Shah, Farooq Rashid, Shuai Wei, Liang Chen, Ge Shan
microRNAs (miRNAs) are critical regulators of gene expression. For elucidating functional roles of miRNAs, it is critical to identify their direct targets. There are debates about whether pulldown of biotinylated miRNA mimics can be used to identify miRNA targets or not. Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays...
October 13, 2017: RNA Biology
https://www.readbyqxmd.com/read/29017520/the-nuclear-receptor-er%C3%AE-engages-ago2-in-regulation-of-gene-transcription-rna-splicing-and-risc-loading
#7
Roberta Tarallo, Giorgio Giurato, Giuseppina Bruno, Maria Ravo, Francesca Rizzo, Annamaria Salvati, Luca Ricciardi, Giovanna Marchese, Angela Cordella, Teresa Rocco, Valerio Gigantino, Biancamaria Pierri, Giovanni Cimmino, Luciano Milanesi, Concetta Ambrosino, Tuula A Nyman, Giovanni Nassa, Alessandro Weisz
BACKGROUND: The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease...
October 6, 2017: Genome Biology
https://www.readbyqxmd.com/read/28977639/biochemical-and-single-molecule-analyses-of-the-rna-silencing-suppressing-activity-of-crpv-1a
#8
Mariko Watanabe, Hiro-Oki Iwakawa, Hisashi Tadakuma, Yukihide Tomari
Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts' RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)-the core of the RNA-induced silencing complex (RISC)-in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC...
August 24, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28977573/dicer-independent-processing-of-small-rna-duplexes-mechanistic-insights-and-applications
#9
Elena Herrera-Carrillo, Ben Berkhout
MicroRNAs (miRNAs) play a pivotal role in the regulation of cellular gene expression via the conserved RNA interference (RNAi) mechanism. Biogenesis of the unusual miR-451 does not require Dicer. This molecule is instead processed by the Argonaute 2 (Ago2) enzyme. Similarly, unconventional short hairpin RNA (shRNA) molecules have been designed as miR-451 mimics that rely exclusively on Ago2 for maturation. We will review recent progress made in the understanding of this alternative processing route. Next, we describe different Dicer-independent shRNA designs that have been developed and discuss their therapeutic advantages and disadvantages...
August 31, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28973861/ctcf-prevents-genomic-instability-by-promoting-homologous-recombination-directed-dna-double-strand-break-repair
#10
Fengchao Lang, Xin Li, Wenhai Zheng, Zhuoran Li, Danfeng Lu, Guijun Chen, Daohua Gong, Liping Yang, Jinlin Fu, Peng Shi, Jumin Zhou
CTCF is an essential epigenetic regulator mediating chromatin insulation, long-range regulatory interactions, and the organization of large topological domains in the nucleus. Phenotypes of CTCF haploinsufficient mutations in humans, knockout in mice, and depletion in cells are often consistent with impaired genome stability, but a role of CTCF in genome maintenance has not been fully investigated. Here, we report that CTCF maintains genome stability, is recruited to sites of DNA damage, and promotes homologous recombination repair of DNA double-strand breaks (DSBs)...
September 25, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28971854/in-vitro-reconstitution-of-chaperone-mediated-human-risc-assembly
#11
Ken Naruse, Eriko Matsuura-Suzuki, Mariko Watanabe, Shintaro Iwasaki, Yukihide Tomari
To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and 5 chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and 5 recombinant chaperone proteins: Hsp90β, Hsc70, Hop, Dnaja2, and p23...
October 2, 2017: RNA
https://www.readbyqxmd.com/read/28964783/nudt21-regulates-3-utr-length-and-microrna-mediated-gene-silencing-in-hepatocellular-carcinoma
#12
Meng Sun, Junyao Ding, Dali Li, Guoping Yang, Zeneng Cheng, Qubo Zhu
Recent studies have shown that several microRNAs (miRNAs) are involved in hepatocellular carcinoma (HCC) tumorigenesis and metastasis; however, the mechanisms responsible for the differences in the functions of these miRNAs in liver cancer remain a mystery. In our previous study, we identified NUDT21 as an interaction partner of argonaute 2 (AGO2). NUDT21 has been reported to be involved in alternative polyadenylation (APA); thus, the interaction between NUDT21 and AGO2 may be a key component of the crosstalk between APA and miRNA-mediated gene silencing in HCC...
September 28, 2017: Cancer Letters
https://www.readbyqxmd.com/read/28942753/microrna-expression-profile-in-raw264%C3%A2-7-macrophage-cells-exposed-to-echinococcus-multilocularis-metacestodes
#13
Xiaola Guo, Yadong Zheng
MicroRNAs (miRNAs) are short noncoding RNAs, involved in the regulation of parasite diseases. However, a role of miRNAs in Echinococcus multilocularis infection remains largely unknown. In this study, we first found the expression levels of key genes involved in miRNA biogenesis and function, including Ago2, Xpo5, Tarbp2 and DgcR8, were obviously altered in the macrophage RAW264·7 cells exposed to E. multilocularis metacestodes. Compared with the control, 18 and 32 known miRNAs were found to be differentially expressed (P 2) in the macrophages exposed to E...
September 25, 2017: Parasitology
https://www.readbyqxmd.com/read/28940437/analysis-of-argonaute-2-microrna-complexes-in-ex-vivo-stored-red-blood-cells
#14
Long Vu, Viswanath Ragupathy, Sandhya Kulkarni, Chintamani Atreya
BACKGROUND: Human enucleated mature red blood cells (RBCs) contain both mature microRNAs (miRNAs) and mRNAs, and we have previously correlated RBC storage lesion processes such as eryptosis, adenosine 5'-triphosphate loss, and RBC indices with differentially expressed miRNAs. Here we have characterized Argonaute 2 (AGO2)-miRNA complexes in stored mature RBCs as a first step toward understanding their role, if any. STUDY DESIGN AND METHODS: In this report AGO2-bound miRNAs in mature RBCs isolated from RBCs collected from three different healthy donors and stored for 24 hours at 4 to 6°C were identified by anti-AGO2 immunoprecipitation (IP) followed by next-generation sequencing of the RNA isolated from the IP...
September 20, 2017: Transfusion
https://www.readbyqxmd.com/read/28939659/helix-7-in-argonaute2-shapes-the-microrna-seed-region-for-rapid-target-recognition
#15
Shannon M Klum, Stanley D Chandradoss, Nicole T Schirle, Chirlmin Joo, Ian J MacRae
Argonaute proteins use microRNAs (miRNAs) to identify mRNAs targeted for post-transcriptional repression. Biochemical assays have demonstrated that Argonaute functions by modulating the binding properties of its miRNA guide so that pairing to the seed region is exquisitely fast and accurate. However, the mechanisms used by Argonaute to reshape the binding properties of its small RNA guide remain poorly understood. Here, we identify a structural element, α-helix-7, in human Argonaute2 (Ago2) that is required for speed and fidelity in binding target RNAs...
September 22, 2017: EMBO Journal
https://www.readbyqxmd.com/read/28934506/clip-seq-analysis-of-multi-mapped-reads-discovers-novel-functional-rna-regulatory-sites-in-the-human-transcriptome
#16
Zijun Zhang, Yi Xing
Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation-maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation...
September 19, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28933656/induction-of-rnai-responses-by-short-left-handed-hairpin-rnai-triggers
#17
Jonathan C Hagopian, Alexander S Hamil, Arjen van den Berg, Bryan R Meade, Akiko Eguchi, Caroline Palm-Apergi, Steven F Dowdy
Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses...
October 2017: Nucleic Acid Therapeutics
https://www.readbyqxmd.com/read/28919329/phospholipase-c%C3%AE-interacts-with-cytosolic-partners-to-regulate-cell-proliferation
#18
REVIEW
Suzanne Scarlata, Ashima Singla, Osama Garwain
Phospholipase Cβ (PLCβ) is the main effector of the Gαq signaling pathway relaying different extracellular sensory information to generate intracellular calcium signals. Besides this classic function, we have found that PLCβ plays an important but unknown role in regulating PC12 cell differentiation by interacting with components in the RNA-induced silencing machinery. In trying to understand the role of PLCβ in PC12 cell differentiation, we find that over-expressing PLCβ reduces PC12 cell proliferation while down-regulating PLCβ increases the rate of cell proliferation...
September 9, 2017: Advances in Biological Regulation
https://www.readbyqxmd.com/read/28910400/exosome-mediated-mir-146a-transfer-suppresses-type-i-interferon-response-and-facilitates-ev71-infection
#19
Yuxuan Fu, Li Zhang, Fang Zhang, Ting Tang, Qi Zhou, Chunhong Feng, Yu Jin, Zhiwei Wu
Exosomes can transfer genetic materials between cells. Their roles in viral infections are beginning to be appreciated. Researches have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate recipient's cellular response and result in productive infection of the recipient host. Here, we showed that EV71 infection resulted in upregulated exosome secretion and differential packaging of the viral genomic RNA and miR-146a into exosomes...
September 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28910321/identification-of-transforming-growth-factor-beta-regulated-micrornas-and-the-microrna-targetomes-in-primary-lung-fibroblasts
#20
Jennie Ong, Wim Timens, Vijay Rajendran, Arjan Algra, Avrum Spira, Marc E Lenburg, Joshua D Campbell, Maarten van den Berge, Dirkje S Postma, Anke van den Berg, Joost Kluiver, Corry-Anke Brandsma
BACKGROUND: Lung fibroblasts are involved in extracellular matrix homeostasis, which is mainly regulated by transforming growth factor-beta (TGF-β), and are therefore crucial in lung tissue repair and remodeling. Abnormal repair and remodeling has been observed in lung diseases like COPD. As miRNA levels can be influenced by TGF-β, we hypothesized that TGF-β influences miRNA expression in lung fibroblasts, thereby affecting their function. MATERIALS AND METHODS: We investigated TGF-β1-induced miRNA expression changes in 9 control primary parenchymal lung fibroblasts using miRNA arrays...
2017: PloS One
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