Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. Structure of the guide-strand-containing Argonaute silencing complex. Nature 456, 209-213

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Nature (Impact Factor: 41.46). 09/2008; 456(7219):209-13. DOI: 10.1038/nature07315
Source: PubMed


The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.

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    • "Animal miRNAs bind their targets via guide nucleotides g2–g8 (Lewis et al., 2003; Rajewsky and Socci, 2004; Krek et al., 2005; Lewis et al., 2005; Lim et al., 2005). Argonaute pre-organizes these ''seed'' nucleotides into a conformation favorable for base pairing (Ma et al., 2004; Parker et al., 2005; Wang et al., 2008b; Elkayam et al., 2012; Nakanishi et al., 2012), pre-paying the entropic penalty inherent in base pairing to a target (Parker et al., 2009). Consequently, the seed sequence determines the binding specificity of Argonaute. "
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    ABSTRACT: Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape 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. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell 07/2015; 162(1):84-95. DOI:10.1016/j.cell.2015.06.029 · 32.24 Impact Factor
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    • "As a general rule, the SS and the 3'-proximal part and 3'-overhang of the AS are more amenable to chemical modification; on the contrary the 5' phosphate, the 5' proximal part and central positions of the AS are more sensitive , especially to multiple or bulky modifications (Bramsen and Kjems, 2013). The 5'- phosphate group of the guide strand is required for the binding to AGO2 (Nykanen et al., 2001; Wang et al., 2008), in addition, the initial interactions between the guide strand and target RNA are mediated by the 5' proximal seed region; therefore modifications of these particular regions may interfere with activity (Wang et al., 2008). Oligonucleotides are synthetized using solid phase phosphoroamidite chemistry. "
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    ABSTRACT: RNA interference is a cellular mechanism by which small molecules of double stranded RNA modulate gene ex-pression acting on the concentration and/or availability of a given messenger RNA. Almost 10 years after Fire and Mello received the Nobel Prize for the discovery of this mechanism in flat worms, RNA interference is on the edge of becoming a new class of therapeutics. With various phase III studies underway, the following years will determine whether RNAi-therapeutics can rise up to the challenge and become mainstream medicines. The present review gives a thorough overview of the current status of this technology focusing on the path to the clinic of this new class of compounds.
    EXCLI Journal 06/2015; 14. · 0.86 Impact Factor
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    • "PIWI and other Argonaute proteins can catalyze endonucleolytic cleavage of their targets. Target cleavage requires extensive complementarity between the guide and the target to enable a conformational change that brings the target closer to the Argonaute active site (Wang et al., 2008, 2009). "
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    ABSTRACT: PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the "Ping-Pong" pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11-the nucleotides paired to piRNA guide positions g10 and g11-the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. We find that 10A is not caused by 1U. Instead, fly Aubergine 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; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3. Copyright © 2014 Elsevier Inc. All rights reserved.
    Molecular Cell 12/2014; 56(5):708. DOI:10.1016/j.molcel.2014.10.016 · 14.02 Impact Factor
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