Figure 5.

Domain mapping of human Drosha. (A) Schematic representation of Drosha mutants. Results from the pri-miRNA processing assay and the DGCR8 binding assay are summarized at the right. (B) Western blot analysis of the mutant proteins. Expression plasmids were transfected into HEK293T cells. Forty-eight hours post-transfection, total cell extract was analyzed by Western blot analysis using anti-Flag antibody. (C) In vitro processing of pri-let-7a-1. The substrate was incubated with wild-type (WT) Drosha or mutant proteins that were prepared by immunoprecipitation in Buffer D-K′100 from transiently transfected HEK293T cells. (D) In vitro binding assay. Drosha protein (wild type [WT] or mutants) were immobilized on anti-Flag beads and incubated with radiolabeled DGCR8 that had been prepared in TnT-coupled reticulocyte lysate system (Promega). Drosha and DGCR8 were incubated with rotation in buffer D-K′250 for 90 min at 4°C.