Recognition of siRNA asymmetry by TAR RNA binding protein.

RNA interference (RNAi) is a means of enacting specific silencing of the expression of a target gene (1, 2). The pathway can be initiated by long double-stranded RNAs (dsRNAs) or pre-microRNAs (pre-miRNAs) processed by the ribonuclease III family enzyme Dicer into 21–27 nucleotide (nt) long, double-stranded small interfering RNAs (siRNAs) or miRNAs with 5′-phosphates and 3′-dinucleotide overhangs (3). Alternatively, siRNAs with the proper structure can be added directly to cells for initiation of the pathway (4, 5). In both cases, silencing is initiated through the RNA induced silencing complex (RISC) and its predecessor, the RISC loading complex (RLC) (6, 7). The RLC is minimally comprised of Dicer, the TAR RNA binding protein (TRBP), and Argonaute 2 (Ago2), although several other proteins have been shown to associate with the RLC and RISC in vivo (8–14). Ago2 is the catalytic component of RISC responsible for degradation of the complementary passenger siRNA strand for generation of active RISC and cleavage of the target mRNA for silencing (15–20). Though it has no enzymatic contribution to siRNA-initiated RNAi, Dicer has been shown to contribute to siRNA processing through coupling dsRNA processing to guide strand loading onto Ago2 (21–23), a role supported by the structure of the RLC in vitro (24). While the same structural studies suggested that TRBP can stabilize the RLC (24), a precise functional contribution of TRBP to siRNA processing has yet to be confirmed. TRBP was first identified by its ability to bind the TAR RNA structure present in human immunodeficiency virus (HIV-1) transcripts (25). Later, it was shown that TRBP can inhibit Protein Kinase R (PKR), an important contributor to the innate response to viral infection (26). Most recently, it was found that TRBP interacts directly with Dicer, as well as the activator of PKR (PACT), through the Medipal domain present at the C-terminus of TRBP (27), and that the interaction is not mediated solely by dsRNA (9, 28). A similar interaction is detected in Drosophila, where Dicer-2 associates with the dsRNA-binding protein (dsRBP) R2D2 (29). Together, the Dicer-2/R2D2 complex senses the relative thermodynamic asymmetry within an siRNA (7). R2D2 binds to the more stable (dsRNA-like) end, leaving Dicer-2 to bind the opposite end. Furthermore, binding is enhanced by the presence of a 5′-phosphate on the passenger strand; hydroxyl groups, such as those present during chemical siRNA synthesis, inhibit binding (7). As a result of the asymmetric binding, the guide strand is preferentially loaded into active RISC, and the passenger strand is degraded (17, 20). Extensive data have shown that human RISC senses asymmetry between the ends of the siRNA or miRNA:miRNA* duplexes, leading to the preferential incorporation of one strand as the guide strand of active RISC (6, 13, 30, 31). This has been accomplished with crude cell extracts and immunopurified proteins (6, 13), and more recently using only recombinant Dicer, TRBP, and Ago2 in a 1:1:1 stoichiometry (10). Asymmetric strand loading by human RISC is also demonstrated in analyses of large siRNA or miRNA datasets (30, 31) and is currently the cornerstone for predicting siRNA efficacy (32, 33). However, unlike in Drosophila, no studies using human cells or proteins have conclusively shown how siRNA asymmetry is detected. Since Ago2 alone cannot bind and select the siRNA guide strand (19), it suggests that its partners in the RLC, Dicer and/or TRBP, contribute to initial siRNA loading, even though other factors such as RNA helicase A (RHA) or PACT may also have an effect in vivo (11, 12, 14). We were interested in characterizing the binding of siRNAs by recombinant TRBP in vitro to determine whether TRBP could sense asymmetry in siRNAs. Our results show that TRBP binds siRNAs and not the analogous siDNAs or siDNA/RNA hybrids. Furthermore, we demonstrate that TRBP alone can sense the asymmetry of an siRNA duplex, thus establishing a key functional role for TRBP in the human RNAi pathway. Additionally, TRBP bound 21 nt ssRNAs in a manner that reflected the asymmetry detected in their corresponding siRNA duplex, providing an indication that TRBP-RNA binding may also be sequence dependent.