Alternative RISC assembly: Binding and repression of microRNA–mRNA duplexes by human Ago proteins

Maja M. Janas; Bingbing Wang; Abigail S. Harris; Mike Aguiar; Jonathan M. Shaffer; Yerramilli V.B.K. Subrahmanyam; Mark A. Behlke; Kai W. Wucherpfennig; Steven P. Gygi; Etienne Gagnon; Carl D. Novina

doi:10.1261/rna.035675.112

Alternative RISC assembly: Binding and repression of microRNA–mRNA duplexes by human Ago proteins

¹Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA
²Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, USA
³Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02141, USA
⁴Qiagen, Inc., Frederick, Maryland 21703, USA
⁵Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
⁶Integrated DNA Technologies, Coralville, Iowa 52241, USA

↵7 Present address: Novartis Institutes for BioMedical Research, Inc., Cambridge, MA 02139, USA
↵8 Present address: Division of Maternal-Fetal Medicine, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA
↵9 Present address: Institute for Research in Immunology and Cancer, Department of Microbiology and Immunology, Montreal University, Montreal, Quebec H3T1J4, Canada

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate protein output from the majority of human mRNAs. In contrast to the consensus view that all miRNAs are associated with Argonaute (Ago) proteins, we determine that miRNAs are expressed in a 13-fold excess relative to Agos in HeLa cells and that miRNAs are bound to mRNAs in a sevenfold excess relative to Agos, implying the existence of miRNA–mRNA duplexes not stoichiometrically bound by Agos. We show that all four human Agos can repress miRNA–mRNA duplexes, but only Ago2 can cleave small interfering RNA–mRNA duplexes in vitro. We visualize direct Ago binding to miRNA–mRNA duplexes in live cells using fluorescence lifetime imaging microscopy. In contrast to the consensus view that Agos bind miRNA duplexes, these data demonstrate that Agos can bind and repress miRNA–mRNA duplexes and support a model of catalytic Ago function in translational repression.

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Footnotes

↵10 Corresponding authors

E-mail etienne.gagnon{at}umontreal.ca

E-mail carl_novina{at}dfci.harvard.edu
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.035675.112.