FIGURE 4.

Dicer’s dsRBD contains a basic region critical for NLS activity. (A) Recombinant GST-dsRBD (wt) was immobilized on GSH-sepharose beads and mixed with the indicated purified factors (Impβ, Imp7) added to E. coli lysate. Bound proteins were eluted and separated by SDS-PAGE followed by Coomassie staining. (B) Representation of the basic patch in Dicer’s dsRBD (blue, with positions of mutated residues indicated). Mutation of its amino acid residues to alanines destroyed the dsRBD NLS activity. Mutation of two additional basic residues, K1887 and K1889, to alanines had no appreciable effect on nuclear localization of the dsRBD-PK reporter (data not shown). (C) Binding of Impβ and Imp7 to Dicer’s dsRBD is dependent on the integrity of the basic patch. The experiment was performed as described in A, comparing Impβ and Imp7 recruitment to the wt and NLSmut dsRBD. (D) HeLa cells were transiently transfected with dsRBD-PK-myc or dsRBD-PK-myc NLSmut (K1891A/R1907A/R1910A). Twenty-four hours post-transfection, cells were fixed and immunofluorescence (IF) was performed with an α-myc antibody. Representative images are shown from a minimum of three independent experiments. Bar, 20 μm.