Endogenous pri-miRNAs that are efficiently processed are enriched in chromatin. (A) Schematic of endogenous pri-miRNAs. Exons are represented as gray boxes; hairpins indicate miRNA locations. Arrowheads indicate the location of primers used for PCR in B, D, and F. Promoters for EBNA (Cp, Wp, and Qp) and BHRF1 (BHRF1p) transcripts are represented by arrows. Binding sites for p53 (p53 BS) are indicated. Schematics are not shown to scale. (B) Nuclear fractionation of endogenous pri-miRNAs. HeLa or U2OS cells were fractionated, RNA was reverse transcribed, and endogenous pri-miRNAs were amplified by PCR using primers diagramed in A from the released, nucleoplasmic supernatant (S; lanes 1 and 3) and the chromatin-associated pellet (P; lanes 2 and 4). β-globin pre-mRNA, β-globin fully spliced mRNA, and TAFII30 fully spliced mRNA served as controls for fractionation efficiency. (C) Northern blot analysis of primary and mature miR-34a expression in HeLa cells after UV irradiation. (D) Nuclear fractionation of HeLa cells 8 h after UV irradiation or mock treatment (–). (E) Northern blot analysis of pri-miR-BHRF1-2∼3 after treatment of Raji or B95-8 cells with DMSO (–) or PMA and Ionomycin (P/I). (F) Nuclear fractionation of Raji cells after 24 h of DMSO (–) or P/I treatment. For all PCRs, the size of the amplified fragment (in base pairs) is indicated at the right of the gel.