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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 level1. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex2,3,4. These pre-miRNAs are cleaved by the RNase III Dicer5,6,7,8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence9. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein10), 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.

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Figure 1: Isolation of a Dicer–TRBP-containing complex.
Figure 2: The Dicer–TRBP complex processes miRNAs and associates with siRNA to form a ternary complex with Ago2.
Figure 3: Stable association of Dicer–TRBP with Ago2.
Figure 4: The Dicer–TRBP complex is required for miRNA biogenesis and post-transcriptional gene silencing.

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We thank T. Beer for assistance with the microcapillary HPLC/mass spectrometry. R.S. was supported by a grant from NIH and American Cancer Society. R.I.G. is a fellow of the Jane Coffin Childs Fund for Medical Research.

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Correspondence to Ramin Shiekhattar.

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Chendrimada, T., Gregory, R., Kumaraswamy, E. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005).

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