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Structural determinants of miRNAs for RISC loading and slicer-independent unwinding
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Structural determinants of miRNAs for RISC loading and slicer-independent unwinding
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Journal Article
Structural determinants of miRNAs for RISC loading and slicer-independent unwinding
2009
Overview
miRNAs are loaded onto Argonautes (Agos) to guide silencing of targets, but duplex unwinding is required for targeting. Detection of
Drosophila
Ago1 complexes containing the duplexed or unwound miRNA now give insight into the basis for cleavage-independent unwinding of miRNA duplexes to generate a functional, mature complex.
MicroRNAs (miRNAs) regulate expression of their target mRNAs through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) family protein as a core component. In
Drosophila melanogaster
, miRNAs are generally sorted into Ago1-containing RISC (Ago1-RISC). We established a native gel system that can biochemically dissect the Ago1-RISC assembly pathway. We found that miRNA-miRNA* duplexes are loaded into Ago1 as double-stranded RNAs in an ATP-dependent fashion. In contrast, unexpectedly, unwinding of miRNA-miRNA* duplexes is a passive process that does not require ATP or slicer activity of Ago1. Central mismatches direct miRNA-miRNA* duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strand positions 12–15 promote conversion of pre-Ago1-RISC into mature Ago1-RISC. Our findings show that unwinding of miRNAs is a precise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
/ Adenosine Triphosphate - pharmacology
/ Animals
/ ATP
/ Biomedical and Life Sciences
/ Drosophila melanogaster - genetics
/ Drosophila melanogaster - metabolism
/ Drosophila Proteins - genetics
/ Drosophila Proteins - metabolism
/ Eukaryotic Initiation Factors
/ Insects
/ Nucleic Acid Conformation - drug effects
/ Proteins
/ Ribonuclease III - metabolism
/ RNA
