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. Author manuscript; available in PMC: 2011 Aug 13.
Published in final edited form as: Mol Cell. 2010 Aug 13;39(3):373–384. doi: 10.1016/j.molcel.2010.07.011

Fig.2. The R-SBE sequence is essential for Smad-mediated regulation of Drosha processing.

Fig.2

A. Schematic diagram of pre-miR-21 wild type and mutant sequences. Red and blue characters indicate the mature miRNA sequence and the R-SBE sequence found in pre-miR-21, respectively. Yellow highlighted characters indicate mutations introduced. B. Mouse C3H10T1/2 cells were transfected with human pri-miR-21 expression constructs, followed by treatment with or without 3 nM BMP4 for 2 hr and subjected to qRT-PCR analysis using primers to detect exogenous pri-miR-21 or pre-miR-21, normalized to GAPDH. Fold induction by BMP relative to mock treated cells is displayed. Induction of WT, 5' mut, and Loop mut by BMP4 is statistically significant. (*P<0.05, n=3) C. Cos7 cells were transfected with pri-miR-21 expression constructs along with Flag-Smad1, Flag-Drosha or Flag-DGCR8 expression constructs, and stimulated with BMP4 for 2 hr. RNA-IP assay using anti-Flag antibody was performed, followed by PCR amplification of exogenous pri-miR-21. Relative enrichment above a non-specific control IgG antibody IP control is shown. Expression of pri- or pre-miR-21 prior to IP was examined by qRT-PCR analysis using indicated primers, normalized to GAPDH (Input). (*P<0.05, n=3) Error bars represent s.e.m. See also supplemental Fig. S1.