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(2-4). These pre-miRNAs are cleaved by the RNase III Dicer(5-8) 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.