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. Author manuscript; available in PMC: 2012 Sep 16.
Published in final edited form as: Mol Cell. 2011 Sep 16;43(6):982–992. doi: 10.1016/j.molcel.2011.07.025

Figure 4. HVS does not utilize the Microprocessor complex.

Figure 4

(A) Northern blot analysis of the expression of hvsA-miR-HSUR4-5p, miR-142-3p, and EBER1 in 293T cells treated with a control siRNA (siCtrl) or a siRNA specific for DGCR8 (siDGCR8) for 48 hr, followed by co-transfection with siDGCR8 and three plasmids expressing HSUR4 and downstream miRNAs, miR-142, or EBER1. U6 provides a loading control. Western blots (lower right two panels) show the knockdown of DGCR8 (a cross-reacting band is marked by an asterisk) with GAPDH as a loading control. (B) 32P-labeled T7-transcribed RNA substrates corresponding to pri-miR-16-2 or to variants of the primary transcript for HSUR4 and its linked miRNAs were incubated with buffer alone (lanes 1-4) or immunoprecipitates containing FLAG-Drosha and FLAG-DGCR8 (lanes 5-8) expressed in 293T cells. Cleavage products were separated on a denaturing polyacrylamide gel. Pri-miRNA substrates are diagramed on the right, with the pre-miRNA hairpin in gray and the box representing the HSUR4-3′ box.