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. 2004 Dec 15;18(24):3016–3027. doi: 10.1101/gad.1262504

Figure 4.

Figure 4.

Role of human DGCR8 in pri-miRNA processing. (A) Immunoprecipitation of the Drosha complex with antibody against Flag. Drosha-Flag protein was expressed in HEK293T cells by transfection of pCK-Drosha-Flag. Total cell extract was prepared from four 10-cm dishes and used for immunoprecipitation with anti-Flag antibody-conjugated agarose beads. (Lane 1) Extract prepared from the same amount of “mock” transfected cells was used as a control. Proteins were separated on 7.5% denaturing polyacrylamide gels and visualized by silver staining. The specific bands were analyzed by MALDI-TOF mass spectrometry. Two forms of truncated Drosha proteins were marked with one or two asterisks (Drosha★ and Drosha★★). Heavy and light chains of anti-Flag antibody are abbreviated as h.c. and l.c. The position of the molecular mass markers is indicated on the left in kilodaltons. (B) Coimmunoprecipitation of DGCR8 and Drosha. V5-tagged DGCR8 and Flag-tagged Drosha proteins were coexpressed in HEK293T cells. Flag-pCK is the backbone for Drosha expression and was used here to match the total amount of plasmids in each transfection. Immunoprecipitation was carried out by incubating total cell extract with anti-Flag antibody-conjugated agarose beads in buffer D-K′250. For Western blot analysis, anti-V5 antibody was used to visualize the V5-DGCR8 protein. (C) In vitro processing of pri-let-7a-1 using Drosha-Flag or Flag-DGCR8 immunoprecipitates prepared in buffer D-K′100. (D) Reconstitution of pri-miRNA processing activity using Drosha-Flag immunoprecipitate and recombinant GST-DGCR8 protein. Pri-miRNA processing activity was measured by in vitro processing of pri-let-7a-1. Drosha-Flag immunoprecipitate was prepared by washing with either buffer A (0.5 M NaCl) or buffer B (2.5 M NaCl); 1.5 μg of recombinant proteins (GST or GST-DGCR8) was added to 1.5 μg of the Drosha-Flag immunoprecipitate. GST-DGCR8 (ΔN275) protein contains 276-773 amino acids of human DGCR8. (E) RT-PCR of pri-miRNA. The siRNA duplex against luciferase, Drosha, or DGCR8 was transfected to HeLa cells. After 72 h, total RNA was prepared and used for RT-PCR. (F) Northern blot analysis of miR-21. RNAi was carried out by transfection of siRNA duplexes to HeLa cells as in E. 5S rRNA bands stained with ethidium bromide are presented as a loading control.