Pri-miRNAs containing a stabilizing viral RNA element, the ENE, accumulate to high levels in the nucleus but are not efficiently processed. (A) Schematic of pri-miRNA constructs. CMV and BGH pA denote the CMV promoter and BGH CPA signal. The pri-miRNA is shown in black, with the mature miRNA indicated in red. Red boxes show five copies of the 79-nt ENE in either the forward or reverse complement (r) orientation. Blue lines below the pri-miRNA sequence specify DIG-tailed oligonucleotide probes used for ISH. (B) Northern blot analysis of pri-miRNA processing. Constructs diagramed in A for pri-let-7 (lanes 1–4) or pri-lin-4 (lanes 5–8) were transfected into HeLa cells, and total RNA was analyzed by Northern blotting using either a denaturing formaldehyde-agarose gel to detect pri-miRNAs (top) or a 15% denaturing polyacrylamide gel to visualize the precursor and mature miRNAs (bottom). Pri-miRNA Northern blots were probed for 7SK as a loading control and for the neomycin resistance gene RNA (NeoR) as a transfection efficiency control. Mature miRNA Northern blots were probed for U6 snRNA as a loading control. 18S denotes cross-hybridization with 18S ribosomal RNA. (C) RT-PCR of pri-let-7. RNA from cells transfected as in B was reverse transcribed followed by PCR with primers designed to amplify pri-let-7. β-actin mRNA was amplified for normalization. Lanes 1–3 show samples treated identically to those in lanes 4–6 but without addition of reverse transcriptase (RT). (D) In vitro processing reactions were performed according to Lee et al. (2002) on pri-let-7 (lanes 1–3) and pri-lin-4 (lanes 4–6) with or without the ENE. Lanes 7–12 show pri-miRNA substrates not incubated in cell extract. Bands marked with asterisks represent processing intermediates or nonspecific degradation products. DNA size markers are in lane 13. (E) Localization of pri-miRNAs in transfected HeLa cells was assessed by ISH using DIG-labeled probes (A, blue lines). Bar, 10 μm.