1. The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA
2. *These authors contributed equally to this work
3. †Present address: The Children's Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, Pennsylvania 19014, USA

Correspondence to: Ramin Shiekhattar¹ (Email: shiekhattar@wistar.org).

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 level¹. 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 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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