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TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level 1 . Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex 2,3,4 . These pre-miRNAs are cleaved by the RNase III Dicer 5,6,7,8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence 9 . Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein 10 ), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer–TRBP with Argonaute 2 (Ago2) 11,12 , the catalytic engine of RISC. The physical association of Dicer–TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer–TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer–TRBP complex not only in miRNA processing but also as a platform for RISC assembly. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Springer Journals

TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

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References (20)

Publisher
Springer Journals
Copyright
Copyright © 2005 by Macmillan Magazines Ltd.
Subject
Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
ISSN
0028-0836
eISSN
1476-4687
DOI
10.1038/nature03868
Publisher site
See Article on Publisher Site

Abstract

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level 1 . Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex 2,3,4 . These pre-miRNAs are cleaved by the RNase III Dicer 5,6,7,8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence 9 . Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein 10 ), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer–TRBP with Argonaute 2 (Ago2) 11,12 , the catalytic engine of RISC. The physical association of Dicer–TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer–TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer–TRBP complex not only in miRNA processing but also as a platform for RISC assembly.

Journal

NatureSpringer Journals

Published: Jun 22, 2005

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