Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human. RNA interference (RNAi)2 is an evolutionarily conserved mechanism for gene silencing mediated through small RNAs of ϳ22 nucleotides in length (1). At least two classes of small RNAs have been described in mammals, micro-RNAs (miRNA) produced from hairpin precursors and small interfering RNAs (siRNAs) derived from long double-stranded RNAs (dsRNAs) (2). Both miRNAs and siRNAs are generated by RNase III-type nuclease Dicer, and they are assembled into effector complex termed RNA-induced silencing complex (RISC) (3, 4). Although two Dicer enzymes Dcr1 and Dcr2 have been found in fruit flies and are responsible for the generation of miRNAs and siRNAs, respectively, there exists only one single Dicer in humans, which produces both miRNAs and siRNAs (3). Dcr2 is known to associate with a dsRNA-binding protein (dsRBP) termed R2D2, which binds to siRNA and facilitates its passage from Dcr2 to RISC (5, 6).A new family of dsRBPs, which includes Loquacious in Drosophila as well as TRBP and PACT in humans, has been shown recently to interact with Dicer and be required for its function in RNAi (7-12). Unlike its counterparts in Drosophila that have only one dsRBP partner, human Dicer appears to associate with two closely related dsRBPs, TRBP and PACT (10 -12). TRBP was originally identified and characterized by its high affinity for TAR, a hairpin RNA encoded by human immunodeficiency virus type 1 (13). PACT was initially cloned as a cellular protein activator of PKR kinase (14, 15). Although both PACT and TRBP bind to PKR and have three similar dsRNA-binding domains (dsRBDs), TRBP exerts an inhibitory effect on PKR (16 -20).Although both TRBP and PACT interact with and support the function of human Dicer in RNAi (10 -12), it is not understood whether their binding with Dicer is simultaneous or mutually exclusive. TRBP and PACT are closely related, and both are capable of homodimerization (21,22). In addition, they share two binding partners, [16][17][...

RIG-I, a virus sensor that triggers innate antiviral response, is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease that mediates RNA interference. Dicer requires double-stranded RNA-binding protein partners, such as PACT, for optimal activity. Here we show that PACT physically binds to the C-terminal repression domain of RIG-I and potently stimulates RIG-I-induced type I interferon production. PACT potentiates the activation of RIG-I by poly(I:C) of intermediate length. PACT also cooperates with RIG-I to sustain the activation of antiviral defense. Depletion of PACT substantially attenuates viral induction of interferons. The activation of RIG-I by PACT does not require double-stranded RNA-dependent protein kinase or Dicer, but is mediated by a direct interaction that leads to stimulation of its ATPase activity. Our findings reveal PACT as an important component in initiating and sustaining the RIG-I-dependent antiviral response.