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https://www.readbyqxmd.com/read/28645918/phosphorylation-of-argonaute-proteins-affects-mrna-binding-and-is-essential-for-microrna-guided-gene-silencing-in%C3%A2-vivo
#1
Miguel Quévillon Huberdeau, Daniela M Zeitler, Judith Hauptmann, Astrid Bruckmann, Lucile Fressigné, Johannes Danner, Sandra Piquet, Nicholas Strieder, Julia C Engelmann, Guillaume Jannot, Rainer Deutzmann, Martin J Simard, Gunter Meister
Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected...
June 23, 2017: EMBO Journal
https://www.readbyqxmd.com/read/28645154/alg-5-is-a-mirna-associated-argonaute-required-for-proper-developmental-timing-in-the-caenorhabditis-elegans-germline
#2
Kristen C Brown, Joshua M Svendsen, Rachel M Tucci, Brooke E Montgomery, Taiowa A Montgomery
Caenorhabditis elegans contains 25 Argonautes, of which, ALG-1 and ALG-2 are known to primarily interact with miRNAs. ALG-5 belongs to the AGO subfamily of Argonautes that includes ALG-1 and ALG-2, but its role in small RNA pathways is unknown. We analyzed by high-throughput sequencing the small RNAs associated with ALG-5, ALG-1 and ALG-2, as well as changes in mRNA expression in alg-5, alg-1 and alg-2 mutants. We show that ALG-5 defines a distinct branch of the miRNA pathway affecting the expression of genes involved in immunity, defense, and development...
June 22, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28642363/nuclear-phosphorylated-dicer-processes-double-stranded-rna-in-response-to-dna-damage
#3
Kaspar Burger, Margarita Schlackow, Martin Potts, Svenja Hester, Shabaz Mohammed, Monika Gullerova
The endoribonuclease Dicer is a key component of the human RNA interference pathway and is known for its role in cytoplasmic microRNA production. Recent findings suggest that noncanonical Dicer generates small noncoding RNA to mediate the DNA damage response (DDR). Here, we show that human Dicer is phosphorylated in the platform-Piwi/Argonaute/Zwille-connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks...
June 22, 2017: Journal of Cell Biology
https://www.readbyqxmd.com/read/28640690/a-new-layer-of-rrna-regulation-by-small-interference-rnas-and-the-nuclear-rnai-pathway
#4
Xufei Zhou, Xiangyang Chen, Yun Wang, Xuezhu Feng, Shouhong Guang
Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process remain poorly understood. For a long time, small ribosomal RNA sequences have been widely treated as non-specific degradation products and neglected as garbage sequences. Recently, we identified a new class of antisense ribosomal siRNAs (risiRNAs) that downregulate pre-rRNA through the nuclear RNAi pathway in C. elegans. risiRNAs exhibit sequence characteristics similar to 22G RNA while complement to 18S and 26S rRNA...
June 22, 2017: RNA Biology
https://www.readbyqxmd.com/read/28639194/rnaa-induced-by-tata-box-targeting-micrornas
#5
Yijun Zhang, Hui Zhang
Recent studies reveal that some nuclear microRNAs (miRNA) and synthesized siRNAs target gene promoters to activate gene transcription (RNAa). Interestingly, our group identified a novel HIV-1-encoded miRNA, miR-H3, which targets specifically the core promoter TATA box of HIV-1 and activates viral gene expression. Depletion of miR-H3 significantly impaired the replication of HIV-1. miR-H3 mimics could activate viruses from CD4(+) T cells isolated from patients receiving suppressive highly active antiretroviral therapy, which is very intriguing for reducing HIV-1 latent reservoir...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/28639188/small-rna-guided-transcriptional-gene-activation-rnaa-in-mammalian-cells
#6
Long-Cheng Li
Small RNA partnering with Argonaute (Ago) proteins plays important roles in diverse biological processes mainly by suppressing the expression of cognate target sequences. Mounting evidence reveals that the small RNA-Ago pathway can also positively regulate gene expression, a phenomenon termed as RNA activation (RNAa), which is evolutionarily conserved from Caenorhabditis elegans to human. In this chapter, I provide a general overview of mammalian RNAa phenomena and their basic characteristics and discuss recent advances toward understanding the nature of the molecular machinery responsible for RNAa and the development of RNAa-based research tools and therapeutics...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/28636937/the-conserved-rna-binding-cyclophilin-rct1-regulates-small-rna-biogenesis-and-splicing-independent-of-heterochromatin-assembly
#7
An-Yun Chang, Stephane E Castel, Evan Ernst, Hyun Soo Kim, Robert A Martienssen
RNAi factors and their catalytic activities are essential for heterochromatin assembly in S. pombe. This has led to the idea that siRNAs can promote H3K9 methylation by recruiting the cryptic loci regulator complex (CLRC), also known as recombination in K complex (RIKC), to the nucleation site. The conserved RNA-binding protein Rct1 (AtCyp59/SIG-7) interacts with splicing factors and RNA polymerase II. Here we show that Rct1 promotes processing of pericentromeric transcripts into siRNAs via the RNA recognition motif...
June 20, 2017: Cell Reports
https://www.readbyqxmd.com/read/28636437/tobacco-rattle-virus-trv-mediated-silencing-of-nicotiana-benthamiana-argonautes-nbagos-reveals-new-antiviral-candidates-and-dominant-effects-of-trv-nbago1
#8
Denis Odokonyero, Maria R Mendoza, Peter Moffett, Herman B Scholthof
The objective of this study was to determine the contribution of different ARGONAUTE proteins in Nicotiana benthamiana (NbAGOs) to the defense against silencing sensitive GFP-expressing viral constructs based on Tomato bushy stunt virus (TBSV) (Tombusvirus), Sunn-hemp mosaic virus (Tobamovirus), and Foxtail mosaic virus (Potexvirus). Upon Tobacco rattle virus (TRV)-mediated down-regulation of NbAGO1, 4, 5, or 6, no effects were noted on susceptibility to any virus construct, whereas knockdown of NbAGO2 specifically prevented silencing of P19-defective TBSV (TGdP19)...
June 21, 2017: Phytopathology
https://www.readbyqxmd.com/read/28634830/the-microrna-effector-rna-induced-silencing-complex-in-hidradenitis-suppurativa-a-significant-dysregulation-within-active-inflammatory-lesions
#9
S Hessam, M Sand, M Skrygan, Falk G Bechara
Recently, we could show that the expression levels of the key regulators of the microRNA (miRNA) maturation and transport were dysregulated in inflamed hidradenitis suppurativa (HS) tissue (Heyam et al. in Wiley Interdiscip Rev RNA 6:271-289, 2015). The RNA-induced silencing complex (RISC) is the central element of the miRNA pathway and regulates miRNA formation and function. We investigated the expression of the RISC components, namely transactivation-responsive RNA-binding protein-1 (TRBP1), TRBP2, protein activator (PACT) of the interferon-induced protein kinase R, Argonaute RISC Catalytic Component-1 (AGO1) and Component-2 (AGO2), metadherin, and staphylococcal nuclease and Tudor domain-containing-1 (SND1) in inflamed HS tissue compared to healthy and psoriatic controls by real-time reverse transcription polymerase chain reaction...
June 20, 2017: Archives of Dermatological Research
https://www.readbyqxmd.com/read/28633390/investigating-microrna-mediated-regulation-of-the-nascent-nuclear-transcripts-in-plants-a-bioinformatics-workflow
#10
Dongliang Yu, Zhonghai Tang, Chaogang Shao, Xiaoxia Ma, Taihe Xiang, Zhihong Fan, Huizhong Wang, Yijun Meng
Most of the microRNAs (miRNAs) play their regulatory roles through posttranscriptional target decay or translational inhibition. For both plants and animals, these regulatory events were previously considered to take place in cytoplasm, as mature miRNAs were observed to be exported to the cytoplasm for Argonaute (AGO) loading and subsequent target binding. Recently, this notion was challenged by increasing pieces of evidence in the animal cells that uncovered the nuclear importation and action of the AGO-associated miRNAs...
June 14, 2017: Briefings in Bioinformatics
https://www.readbyqxmd.com/read/28624536/microrna-dependent-regulation-of-metamorphosis-and-identification-of-micrornas-in-the-red-flour-beetle-tribolium-castaneum
#11
Wei Wu, Wenfeng Xiong, Chengjun Li, Mengfan Zhai, Yao Li, Fei Ma, Bin Li
To date, although some microRNAs (miRNAs) have been discovered in the holometabolism insect Tribolium castaneum, large numbers of miRNAs still require investigation. Knocking down Dicer-1 (Dcr-1) and Argonaute-1 (Ago-1) in late larvae impaired miRNA synthesis, affected the juvenile hormone pathway by up-regulating Methoprene-tolerant (Met) and Krüppel-homolog1 (Kr-h1) transcript levels, and resulted in a series of defects in T. castaneum development and metamorphosis. Thus, high-throughput Illumina/Solexa sequencing was performed with a mixed sample of eight key developmental stages of T...
June 14, 2017: Genomics
https://www.readbyqxmd.com/read/28609472/no-evidence-for-genome-editing-in-mouse-zygotes-and-hek293t-human-cell-line-using-the-dna-guided-natronobacterium-gregoryi-argonaute-ngago
#12
Nay Chi Khin, Jenna L Lowe, Lora M Jensen, Gaetan Burgio
A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5'-phosphorylated guide DNA (gDNA) from 2...
2017: PloS One
https://www.readbyqxmd.com/read/28608350/phylogenetic-and-evolutionary-analysis-of-plant-argonautes
#13
Ravi K Singh, Shree P Pandey
Comparative sequence analysis is widely used for the reconstruction of phylogeny and for understanding the evolutionary history of gene families. Here, we describe the methodologies to reconstruct the phylogenetic and evolutionary history of a gene family across genomes with a focus on the ARGONAUTE (AGO) family of proteins in plants. The method described here may easily be adapted for studying molecular evolution of a wide variety of gene families. We enlist methods as well as parameters for the collection of molecular data (nucleic acids and peptides), preparation of datasets, and selection of evolutionary models and various methods for the phylogenetic and evolutionary analysis, such as maximum likelihood and Bayesian inference...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608349/identification-and-evolutionary-characterization-of-argonaute-binding-platforms
#14
Joshua T Trujillo, Rebecca A Mosher
ARGONAUTE (AGO) proteins are eukaryotic RNA silencing effectors that interact with their binding partners via short peptide motifs known as AGO hooks. AGO hooks tend to cluster in one region of the protein to create an AGO-binding platform. In addition to the presence of AGO hooks, AGO-binding platforms are intrinsically disordered, contain tandem repeat arrays, and have weak sequence conservation even between close relatives. These characteristics make it difficult to identify and perform evolutionary analysis of these regions...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608348/identification-and-analysis-of-wg-gw-argonaute-binding-domains
#15
Andrzej Zielezinski, Wojciech M Karlowski
WG/GW domains recruit ARGONAUTE (AGO) proteins to distinct silencing effector complexes using combinations of just two amino acids: tryptophan (W) and glycine (G), forming a wide arsenal of highly simplified interaction surfaces. These unstructured domains exhibit very low sequence identity and excessive length polymorphism, which makes identification of new AGO-binding proteins a challenging task as they escape detection with standard sequence comparison-based methods (e.g., BLAST, HMMER).In this chapter, we explain the use of tools for prediction of AGO-binding WG/GW domains in protein sequences...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608347/structural-and-functional-characterization-of-plant-argonaute-mid-domains
#16
Filipp Frank, Bhushan Nagar
The interaction of small silencing RNA 5' nucleotides with the MID domain of ARGONAUTE (AGO) proteins provides an anchor point that contributes to strong binding between RNA and protein. The following protocols describe the necessary procedures to characterize the structure of AGO MID domains using X-ray crystallography as well as their interaction with nucleotides that mimic the 5' end of small silencing RNAs using two-dimensional NMR spectroscopy.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608346/computational-analysis-of-genome-wide-argonaute-dependent-dna-methylation-in-plants
#17
Kai Tang, Cheng-Guo Duan, Huiming Zhang, Jian-Kang Zhu
Whole-genome bisulfite sequencing (WGBS) has become a powerful tool to dissect genome-wide methylation profiles at single-base resolution. In this chapter we describe in detail the bioinformatics pipeline used for the analysis of ARGONAUTE-dependent DNA methylation in Arabidopsis thaliana. We provide tools and command lines used for mapping bisulfite sequencing reads, for estimating methylation levels at individual cytosine sites, for identifying differentially methylated regions (DMRs), and for calculating methylation levels of DMRs...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608343/cloning-and-characterization-of-argonaute-genes-in-tomato
#18
Zhiqiang Xian, Fang Yan, Zhengguo Li
Argonaute (AGO) proteins are core elements in plant posttranscriptional RNA silencing pathways. The identification and functional characterization of tomato (Solanum lycopersicum) AGOs will help to better understand RNA silencing-based posttranscriptional regulation in fleshy fruits. Here we describe how to identify and clone SlAGO genes, as well as the methodology for their functional characterization.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608342/argonaute-genes-in-salvia-miltiorrhiza-identification-characterization-and-genetic-transformation
#19
Meizhen Wang, Yuxing Deng, Fenjuan Shao, Miaomiao Liu, Yongqi Pang, Caili Li, Shanfa Lu
Small RNA-mediated gene silencing is a vital regulatory mechanism in eukaryotes that requires ARGONAUTE (AGO) proteins. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant. Therefore, it is important to characterize S. miltiorrhiza AGO family genes as they may be involved in multiple metabolic pathways. This chapter introduces the detailed protocol for SmAGO gene prediction and molecular cloning. In addition, an Agrobacterium-mediated genetic transformation method for S. miltiorrhiza is presented...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28608341/isolation-and-characterization-of-argonaute-mutants-in-chlamydomonas
#20
Tomohito Yamasaki
Random insertional mutagenesis and subsequent reverse genetic screening allow the isolation of mutants of interest. Here I describe the protocol for generating a tag insertion line and subsequent PCR-based screening for ARGONAUTE mutants as an example of a reverse genetic screen for the unicellular green alga Chlamydomonas reinhardtii.
2017: Methods in Molecular Biology
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