Journal
RNA
Volume 18, Issue 11, Pages 2041-2055Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.035675.112
Keywords
Argonautes; fluorescence lifetime imaging microscopy (FLIM); microRNAs; RISC loading
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Funding
- Cancer Research Institute Postdoctoral Fellowship
- Distinguished Young Scholars Award from the W.M. Keck Foundation
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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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