(310f) Recognition of siRNA by TAR RNA Binding Protein (TRBP)

RNA interference (RNAi) provides a powerful means for regulating gene expression. Although it is a relatively recent discovery, it has already proven useful in characterizing the functions of proteins and is the basis for human therapeutics currently in development. For RNAi to realize its full potential, a more complete understanding of the mechanism is required with our particular emphasis being the design and delivery of the initiator molecules of RNAi, small interfering RNA (siRNA).

The recognition of siRNAs by the RNA induced silencing complex (RISC) and its precursor, the RISC loading complex (RLC), is a key step in the RNAi pathway that controls the subsequent sequence-specific mRNA degradation. In Drosophila, selection of the appropriate guide strand has been shown to be mediated by the RLC protein R2D2, which senses the relative hybridization stability between the two ends of the siRNA. A protein with similar function has yet to be conclusively identified in humans, but as a result of empirical evidence from experiments with hundreds of different siRNAs, human RISC is also known to recognize siRNA asymmetry.

We will present results showing that human TAR RNA binding protein (TRBP) alone can bind siRNAs in vitro and sense their asymmetry in the absence of ATP. We will also demonstrate that TRBP can bind 21 nt long single-stranded RNA, though with far lower affinity than for double-stranded siRNA, and that the binding reflects the bias observed with the full siRNA duplex. This suggests that TRBP binding may be both sequence and stability dependent. Finally, our attempts to alter TRBP interactions with siRNA through the inclusion of sequence mismatches, DNA substitutions, or chemical modifications within the siRNA will be described. The results suggest a role for TRBP in the mechanism of RISC formation as well as a means to improve siRNA functionality through modification of the RNA or its sequence. Future efforts to capitalize on our discoveries will also be highlighted.