Biochemical characterization of RDE-4, a Caenorhabditis elegans protein involved in RNA interference.

摘要

RNA interference (RNAi) is a conserved biological response in which exposure to double-stranded RNA (dsRNA) results in the sequence-specific degradation of complementary mRNAs. RNAi is initiated when the RNase III enzyme Dicer cleaves long dsRNA substrates into small interfering RNAs (siRNAs), which later guide mRNA degradation as part of the RNA-induced silencing complex (RISC). In all organisms, accessory dsRNA-binding proteins (dsRBPs) facilitate Dicer functions in RNAi. In C. elegans, the dsRBP RDE-4 is required for the production of siRNAs, but is not required downstream of this step. Likewise, RDE-4 is not required during micro-RNA (miRNA) maturation, in which Dicer processes miRNAs from small (∼22bp) stem-loop precursors.;Attempts at quantifying the level of cooperativity in RDE-4/dsRNA interactions are described in Chapter 3. For comparison, the human dsRBP, TRBP, was cloned, over-expressed and purified, reasoning it would bind noncooperatively since it acts downstream of siRNA production, in facilitating their incorporation into RISC. The quantification studies proved to be unsuccessful, but the theory for the mathematic treatment of binding data and initial binding studies of TRBP are presented.;Lastly, studies presented in Chapter 4 demonstrate that the distinct in vivo roles of RDE-4 and TRBP are reflected in their in vitro binding properties. I show that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds noncooperatively, binding different length dsRNAs with constant affinities. Additionally, I report the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity in rde-4 mutant extracts, which taken together indicate RDE-4 promotes activity using its dsRBM2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation.;How RDE-4 discriminates between long and short dsRNAs was unclear prior to the work described in this dissertation. In Chapter 2, I describe the cloning, over-expression and purification of RDE-4, followed by a detailed description of its dsRNA binding properties. I show that consistent with its in vivo roles, RDE-4 binds long dsRNA with high affinity and has low affinity for siRNA. I report that high affinity binding of long dsRNA by RDE-4 is due to positive cooperativity, and postulate that this may be a general mechanism for how dsRBPs discern dsRNA length. I also report studies indicating that RDE-4 exists as a homodimer and that dimerization is mediated by its C-terminus. Importantly, I show recombinant RDE-4 is able to reconstitute Dicer activity in rde-4 mutant extracts, in a manner dependent on its C-terminus.