The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing

Shang L. Lian; Songqing Li; Grant X. Abadal; Brad A. Pauley; Marvin J. Fritzler; Edward K.L. Chan

doi:10.1261/rna.1229409

The C-terminal half of human Ago2 binds to multiple GW-rich regions of GW182 and requires GW182 to mediate silencing

¹Department of Oral Biology, University of Florida, Gainesville, Florida 32610-0424, USA
²Department of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada

↵3 These authors contributed equally to this work.

Abstract

MicroRNA (miRNA)-mediated silencing is a post-transcriptional mechanism that regulates translation of mRNAs primarily via their 3′-UTR. Ago2 binds miRNA directly and is the core component of miRNA-induced silencing complex. GW182 is another important factor in miRNA-mediated silencing, and its interaction with Ago2 is evolutionarily conserved. However, the GW182-Ago2 interaction in humans has not been characterized thoroughly, and the role of GW182 in the mammalian miRNA pathway remains unclear. In the current study, we generated a set of GST-, green fluorescence protein (GFP)-, or 3xFlag-tagged deletion constructs of GW182 and Ago2 to further analyze GW182-Ago2 interactions. The C-terminal half of Ago2 interacted with four nonoverlapping GW-rich regions of GW182, and this interaction recruited Ago2 to GWB. Furthermore, the interaction with GW182 was observed in all four human Ago proteins. Most interestingly, tethering the C-terminal half of Ago2 to the 3′-UTR of reporter mRNA recapitulated translational repression comparable to that of tethered Ago2, and this repression was greatly impaired upon GW182 knockdown. In comparison, the N-terminal half of Ago2 did not bind GW182 and did not retain the repression function of Ago2. Our data strongly support a model in which Ago2 recruits GW182 to the 3′-UTR of mRNA to mediate silencing, and suggest that GW182 may contribute to enhancement in translational repression by interacting with multiple Ago proteins from multiple miRNA target sites in the same or adjacent 3′UTR.

Keywords:

Keywords

Footnotes

Reprint requests to: Edward K.L. Chan, Department of Oral Biology, University of Florida, Gainesville, FL 32610-0424, USA; e-mail: echan{at}ufl.edu; fax: (352) 273-8829.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FL, firefly luciferase; GFP, green fluorescence protein; GWB, GW/P bodies; IIF, indirect immunofluorescence; NHA, hemagglutinin tag with a 22-amino acid-long N peptide specifically recognizing the BoxB hairpin; PCR, polymerase chain reaction; RL, Renilla luciferase; miRNA, microRNA; nt, nucleotide; siRNA, small interfering.RNA.
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1229409.
- Received June 17, 2008.
- Accepted February 5, 2009.