Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing

  1. Phillip D. Zamore2,4,5
  1. 1Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
  2. 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
  3. 3Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
  4. 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 3′ 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 complementary 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

    Footnotes

    • Received April 14, 2011.
    • Accepted July 14, 2011.

    Freely available online through the RNA Open Access option.

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