Human TRBP and PACT Directly Interact with Each Other and Associate with Dicer to Facilitate the Production of Small Interfering RNA

Abstract

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][...

Protein Components of the microRNA Pathway and Human Diseases

“…The accumulation of mature miRNAs in vivo and the efficiency of siRNA-induced RNAi are affected upon depletion of PACT (74). These findings were confirmed in shRNA-induced RNAi, but not when using siRNAs, suggesting that TRBP and PACT function primarily in siRNA production (75). Moreover, the presence of both TRBP and PACT increased the ability of Dicer to cleave a 566-bp long dsRNA in vitro, as compared to TRBP and PACT added individually (75).…”

“…The C-terminal domain of PACT can also mediate homomultimerization (79). Interestingly, a recent study demonstrated that PACT directly interacts with TRBP and that this complex associates with Dicer to facilitate production of siRNAs (75).…”

Increased virus replication in mammalian cells by blocking intracellular innate defense responses

“…11,13 Moreover, two factors TRBP and PACT shown to be important in mammalian RNAi were in fact first described to have IFN effector functions. [44][45][46][47] Recent evidence indicates that the IFN's antiviral activity is mediated by the miRNA arm of the RNAi pathway. 48 Overall, these overlaps suggest that the IFN system and RNAi pathway may act cooperatively to control virus infection.…”

Emergence of a Complex Relationship between HIV-1 and the microRNA Pathway

“…In fact, PACT can bind directly to TRBP and form a ternary complex with Dicer and TRBP to facilitate the production of siRNAs by Dicer. Knockdown of both TRBP and PACT in cultured mammalian cells led to a significant inhibition of gene silencing mediated by shRNAs, but not by siRNAs, suggesting that TRBP and PACT function primarily at the step of siRNA production (97). Despite exerting opposite effects on PKR, PACT and TRBP may thus play a similar, possibly redundant, role in miRNA biogenesis and function.…”