Abstract
Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Substitution
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Animals
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Argonaute Proteins
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Cell Line
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Eukaryotic Initiation Factor-2 / analysis
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Eukaryotic Initiation Factor-2 / genetics
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Eukaryotic Initiation Factor-2 / metabolism*
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Humans
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Kinetics
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Mice
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Mice, Knockout
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MicroRNAs / metabolism*
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RNA Precursors / metabolism*
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RNA Processing, Post-Transcriptional
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism*
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RNA-Induced Silencing Complex / metabolism
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Recombinant Fusion Proteins / metabolism
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Ribonuclease III / genetics
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Ribonucleoproteins / analysis
Substances
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AGO2 protein, human
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Ago2 protein, mouse
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Argonaute Proteins
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Eukaryotic Initiation Factor-2
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MicroRNAs
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RNA Precursors
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RNA-Binding Proteins
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RNA-Induced Silencing Complex
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Recombinant Fusion Proteins
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Ribonucleoproteins
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Ribonuclease III