Pri-miRNAs transcribed by RNA polymerase III do not localize to nuclear foci. (A) miRNA constructs used for transfection. U6 denotes the RNA polymerase III promoter; the Pol III termination signal consisted of five thymidylate residues (T). (B) ISH was performed as detailed in Fig. 1 E for pri-lin-4 constructs (a and b) or pri-miR-30 constructs (c and d). (C) Northern blot analysis of mature miRNA species produced from transfections with decreasing amounts of CMV-pri-miR-30 (lanes 2–4) or U6-pri-miR-30 (lanes 6–8) constructs compared with empty vector (−; lanes 1 and 5). (D) Cells transfected as in B were fixed, and IF was performed for Drosha followed by ISH as described in Fig. 6 B. Nuclei were stained with DAPI. Bars, 10 μm.