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Argonaute Protein Identity and Pairing Geometry Determine Cooperativity in Mammalian RNA Silencing University of Massachusetts Medical School eScholarship@UMMS GSBS Student Publications Graduate School of Biomedical Sciences 2011-10-01 Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing Jennifer A. Broderick University of Massachusetts Medical School Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_sp Part of the Biochemistry, Biophysics, and Structural Biology Commons, Medicine and Health Sciences Commons, and the Neuroscience and Neurobiology Commons Repository Citation Broderick JA, Salomon WE, Ryder SP, Aronin N, Zamore PD. (2011). Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing. GSBS Student Publications. https://doi.org/10.1261/rna.2778911. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/1768 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Student Publications by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. Downloaded from rnajournal.cshlp.org on February 28, 2012 - Published by Cold Spring Harbor Laboratory Press Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing Jennifer A. Broderick, William E. Salomon, Sean P. Ryder, et al. RNA 2011 17: 1858-1869 originally published online August 30, 2011 Access the most recent version at doi:10.1261/rna.2778911 Supplemental http://rnajournal.cshlp.org/content/suppl/2011/08/03/rna.2778911.DC1.html Material References This article cites 59 articles, 19 of which can be accessed free at: http://rnajournal.cshlp.org/content/17/10/1858.full.html#ref-list-1 Article cited in: http://rnajournal.cshlp.org/content/17/10/1858.full.html#related-urls Open Access Freely available online through the RNA Open Access option. Email alerting Receive free email alerts when new articles cite this article - sign up in the box at the service top right corner of the article or click here To subscribe to RNA go to: http://rnajournal.cshlp.org/subscriptions Copyright © 2011 RNA Society Downloaded from rnajournal.cshlp.org on February 28, 2012 - Published by Cold Spring Harbor Laboratory Press Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing JENNIFER A. BRODERICK,1 WILLIAM E. SALOMON,2 SEAN P. RYDER,2 NEIL ARONIN,3 and PHILLIP D. ZAMORE2,4,5 1Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA 3Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA 4Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA ABSTRACT Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 39 untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially com- plementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins. Keywords: Argonaute; cooperativity; microRNA; miRNA; RNAi; siRNA; RNA silencing INTRODUCTION promotes nucleophilic attack by hydroxide on the phos- phodiester bond (Kanaya et al. 1996; Haruki et al. 2000; In plants and animals, small silencing RNAs such as small Martinez and Tuschl 2004; Schwarz et al. 2004; Song et al. interfering RNAs (siRNAs) and microRNAs (miRNAs) pro- 2004; Rivas et al. 2005). vide the specificity determinants for Argonaute proteins. A The human genome encodes four Argonaute paralogs— small RNA guide bound to an Argonaute protein is called Ago1, Ago2, Ago3, and Ago4—and most cultured mamma- the RNA-induced silencing complex (RISC) (Hammond lian cell lines express all four proteins, albeit in different et al. 2000; Hannon 2002; Du and Zamore 2007; Matranga proportions (Meister et al. 2004). Of the four mammalian and Zamore 2007; Ghildiyal and Zamore 2009); binding of Argonautes, only Ago2 retains the ability to catalyze site- RISC to the 39 untranslated region (UTR) of an mRNA si- specific, small RNA-directed endonucleolytic target cleavage lences its expression (Lee et al. 1993; Wightman et al. 1993; (Liu et al. 2004; Meister et al. 2004). Like Ago2, Ago3 Olsen and Ambros 1999; Lai 2002; Doench et al. 2003; contains an apparent catalytic triad, but unlike Ago2, it lacks Grimson et al. 2007). Argonaute proteins are structural endoribonuclease activity. For Ago1 and Ago4, there is no homologs of RNase H that typically cleave their target RNAs catalytic triad, explaining their lack of endoribonuclease after the nucleotide paired to the tenth base of the small activity (Meister et al. 2004; Rivas et al. 2005; Azuma-Mukai RNA guide (Elbashir et al. 2001a,b; Tolia and Joshua-Tor et al. 2008). Extensive, but not complete, complementarity 2007). Cleavage requires three key amino acids—D, D, between a small RNA guide and an mRNA is required for H—that form a magnesium-binding catalytic triad, which Argonaute-catalyzed target cleavage (Hutva´gner and Zamore 2002; Schwarz et al. 2002; Haley and Zamore 2004; Liu et al. 2004; Meister et al. 2004; Rivas et al. 2005). In contrast, small 5 Corresponding author. RNAs with only partial complementarity to their target E-mail [email protected]. Article published online ahead of print. Article and publication date are mRNAs, especially those bearing mismatches near the cleav- at http://www.rnajournal.org/cgi/doi/10.1261/rna.2778911. age site, cannot direct endonucleolytic cleavage of their 1858 RNA (2011), 17:1858–1869. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2011 RNA Society. Downloaded from rnajournal.cshlp.org on February 28, 2012 - Published by Cold Spring Harbor Laboratory Press Requirements for cooperativity in RNA silencing target (Holen et al. 2002) but instead reduce the stability of RESULTS the target mRNA (Guo et al. 2010) and, in some conditions, cause translational repression (Doench et al. 2003; Doench Experimental paradigm and Sharp 2004). At least three distinct regulatory mechanisms could explain Experimental and computational analyses suggest that a the enhanced silencing of reporter mRNAs containing mul- single miRNA can regulate hundreds of genes, because a tar- tiple miRNA-binding sites (Fig. 1). A cooperative binding get mRNA need only pair with the seed sequence of a small model posits that the binding of a miRNA:Argonaute pro- RNA—comprising nucleotides 2 through 7 or 8—to recruit tein complex to one site increases the affinity of a second RISC and promote repression (Lewis et al. 2003; Doench miRNA:Argonaute complex for an adjacent site (Fig. 1A). and Sharp 2004; Rajewsky and Socci 2004; Brennecke In this model, the binding of the first bulged siRNA would A et al. 2005; Krek et al. 2005; Lewis et al. 2005; Lim et al. have a higher dissociation constant, KD, than subsequent B C 2005; Grimson et al. 2007; Baek et al. 2008; Selbach et al. binding events, KD and KD; we predict that the amount of 2008; Friedman et al. 2009). Multiple, partially comple- siRNA required to silence a reporter would decrease with mentary small RNAs, when bound to the 39 UTR of a an increasing number of target sites as cooperativity be- luciferase reporter target mRNA, may function cooperatively tween bound Argonautes increases. Such cooperativity in to repress its translation (Doench et al. 2003; Bartel and small RNA-directed silencing might arise from direct Chen 2004), and most mRNAs contain multiple potential interactions between adjacent Argonaute proteins. Alterna- miRNA-binding sites in their 39 UTRs (Lee et al. 1993; tively, a pair of Argonaute proteins might be bridged by Wightman et al. 1993; Reinhart et al. 2000; Abrahante one or more additional proteins. In a cooperative function et al. 2003; Lin et al. 2003; Bartel 2004; Grimson et al. 2007; Friedman et al. 2009). However, the molecular basis for cooperativity in small RNA silencing remains unknown. Here, we show that both the nature of siRNA:mRNA target pairing and the identity of the Argonaute protein to which the small RNA is bound deter- mine whether multiple target sites act cooperatively to recruit RISC. Small RNAs that pair perfectly to multiple target sites silenced noncooperatively when the small RNA guide acts through Ago2, whereas silencing directed by either perfectly or imperfectly pairing small RNAs bound to Ago1, Ago3, or Ago4 acted cooperatively to silence mRNA bearing multiple small-RNA- binding sites. Cooperativity required
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