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Abstract
Double-stranded RNA (dsRNA)-binding proteins facilitate Dicer functions in RNA interference.
Caenorhabditis elegans RDE-4 facilitates cleavage of long dsRNA to small interfering
RNA (siRNA), while human trans-activation response RNA-binding protein (TRBP) functions
downstream to pass siRNA to the RNA-induced silencing complex. We show that these
distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially
binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA
length. These properties are mechanistically based on the fact that RDE-4 binds cooperatively,
via contributions from multiple domains, while TRBP binds noncooperatively. Our studies
offer a paradigm for how dsRNA-binding proteins, which are not sequence specific,
discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs
and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity
using its dsRNA-binding motif 2 to bind dsRNA, its linker region to interact with
Dicer, and its C-terminus for Dicer activation.