Elucidating the role of TRBP in RNA interference

TRBP (HIV-1 transactivating response RNA binding protein) is part of the minimal RISC (RNA-induced silencing complex) loading complex (RLC) which consists of Dicer, Argonaute (Ago) and TRBP. TRBP is a dsRNA binding protein encompassing two dsRNA binding domains (dsRBD) and a C-terminal Medipal domain which plays a role in protein-protein interactions. The dsRBDs interact with the minor groove of RNA duplexes in a sequence-independent manner. Following dsRNA binding, TRBP can form multimeric complexes. Binding of siRNA to RISC, followed by the release of the passenger strand, are important steps during initiation of RNAi. However, so far the mechanistic details remain elusive. Several experimental data strongly suggest that TRBP plays an important role most probably mediated via its ability to bind dsRNA, although this remains to be fully elucidated. In this work we report about biochemical and pre-steady-state kinetic characterization of TRBP/nucleic acid interactions. Performing fluorescence measurements using labeled RNAs, we were able to determine the equilibrium dissociation constant (K_d) for single and double stranded siRNAs. In a next set of experiments, we are about to determine the mechanism of TRBP nucleic acid binding applying pre-steady-state techniques. In parallel, the effect of TRBP on Ago2-mediated mRNA cleavage is investigated. Our final goal is to understand the function of TRBP during siRNA-mediated down-regulation of target mRNAs.