SYBR green master mix (Bio-Rad) and primers speciﬁc for human Dicer or the internal control gene, human acidic ribosomal phosphor-protein P0 (RPLP0). The PCR and ﬂuorescence measurement was performed in a Bio-Rad iCycler using the following cycling conditions: 958C for 9 min, followed by 40 cycles to 3-step PCR with 958C for 30 s, 608C for 30 s and 728C for 30 s. Dicer expression was normalized to levels of the RPLP0 internal control gene. All data points were performed in triplicate and the experiment was performed twice. PCR primer sequences for the RT-PCR analysis are available from the authors on request. For immunoblot analysis, whole-cell extracts were col- lected from wild-type and Dicer mHelicase cells and HEK293 cells (CRL-1573) using the MPer reagent accord- ing to the manufacturer’s instructions (Pierce) and the protein concentration determined by Bradford analysis. Thirty micrograms of protein was separated on a 10% resolving SDS/PAGE gel, transferred to PVDF, blocked in 5% non-fat dried milk in TBST, and incubated with the following Dicer antibodies: N-terminus (Abcam, clone 13D6, 1:300 dilution) or C-terminus (Santa Cruz Bio- technologies, clone H-212, 1:200 dilution), followed by an incubation with HRP-labeled secondary antibodies and detection by ECL chemiluminescence. Separate parts of the membrane were incubated with primary anti- bodies speciﬁc to beta-actin and detected with secondary antibodies and HRP staining as described above for Dicer. Homology modeling of the helicase insertion was carried out using the HEF helicase from Pyrococcus furiosus (PDB ﬁle 1WP9) as a template for visualizing the helicase domain of human Dicer since this domain of Dicer has not been crystallized (26). The sequences were aligned using Figure 1 in Nishino et al. as a guide (26). The program MODELLER 9.0 (27) running within Discovery Studio 2.0 (Accelrys, San Diego, CA) was used to build a model of HEF helicase with a 43-residue loop corresponding to the in-frame insertion mutation spliced into motif II. Luciferase assays For miRNA assays, wild-type and Dicer mHelicase cells were transfected in 24-well dishes using Lipofectamine 2000 (LF2000) according to the manufacturer’s directions. Each well received the following: 12.5 ng of psi-Check-93 target plasmid, 137.5 ng of miRNA expression plasmid (either MCM7 or the irrelevant control HIV) and 150 ng of pcDNA3.1 vector (either empty vector or wild-type Dicer cDNA), and 0.75 ml of LF2000. Twenty-four hours post-transfection, luciferase activity was determined using the Dual Luciferase system (Promega). For sh- and lhRNA assays, wild-type and Dicer mHelicase mutant cells were transfected in 24-well dishes using LF2000. Each well received the following: 100 ng of the psiCheck target plasmid and 1–100 ng of the sh- or lhRNA expression plasmid. In experiments where 1 ng of sh- or lhRNA expression vector was used, the total amount of plasmid DNA was increased to 200 ng by adding the U6 empty vector. In experiments targeting the HIV genome, each well received 100 ng of pNL4- 3.Luc target plasmid, 100 ng of sh or lhRNA expression Nucleic Acids Research, 2008, Vol. 36, No. 20 6513 and 1 ng of a Renilla luciferase expression plasmid (Promega). Twenty-four hours post-transfection, lucifer- ase activity was analyzed using the Dual Luciferase system (Promega). Northern blot analysis For miRNA expression analysis, either total RNA or small RNAs were collected from wild-type or Dicer mHelicase cells using RNA STAT-60 (Tel-Test) (total RNA) or the miRVANA miRNA isolation kit according to the manufacturer’s instructions (Ambion). Ten micro- grams of total RNA or 2 mg of miRVANA RNA were separated in a 15%/8 M urea denaturing polyacrylamide gel, electro-transferred to a Hybond-N+ membrane (Amersham Bioscience), and probed with a 32P-labeled oligonucleotide that is complementary to the mature miR-21 and consists of 20-O-methyl-modiﬁed RNA bases. For the detection of miR-23a and miR-27a, 32P-labeled DNA oligonucleotides that are complemen- tary to the mature miRNA were used as probes for hybri- dization. Following successive washes of increasing stringency, the blot was analyzed by autoradiography. For sh- and lhRNA expression analyses, total RNA was extracted and puriﬁed with RNA STAT-60 (Tel-Test) according to the manufacturer’s instructions. Twenty micrograms of total RNA were separated in an 8% poly- acrylamide denaturing gel, electro-transferred to a Hybond-N membrane and probed with 32P-labeled syn- thetic oligonucleotides complementary to either sense or antisense sequences of the speciﬁc siRNA. Following suc- cessive washes of increasing stringency, the blot was ana- lyzed by autoradiography. The processing eﬃciency for each hairpin RNA (Probe 1, Probe 2, Probe 3) was determined by autoradiography and dosimetry as follows: Processing eﬃciency = matureIDV/(matureIDV + precursorIDV). IDV, Integrated Density Value. In vitro processing analysis Preparation of cell extracts and in vitro processing were performed as described elsewhere, with some modiﬁca- tions (28). Brieﬂy, 1 107 cells (wild type and mHelicase) were washed twice with ice-cold PBS, resus- pended in the following buﬀer: 20 mM HEPES-KOH pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 5% glycerol and protease inhibitor (Complete Mini, Roche), and lysed by sonication. Cell extracts were cleared by cen- trifugation at 13 000 g for 15 min at 48C and then quickly frozen in an ethanol:dry ice bath. Protein concentrations of the cell extracts were determined by the Bradford assay. For in vitro analysis of pre-miRNA, a synthetic RNA with the sequence (50-UAGCUUAUCAGACUGAUG UUGACUGUUGAAUCUCAUGGCAACACCAGUC GAUGGGCUGUC-30) was 50-end labeled with 32P-g- ATP and the full-length RNA was gel-puriﬁed and pre- cipitated with ethanol by standard methods after labeling. The labeled RNA was heated to 958C, 3 min, followed by 758C, 3 min, and then removed from the heat to cool to room temperature to allow the RNA to fold into the proper hairpin structure. For in vitro reactions using recombinant human Dicer (rDicer), 5 104 c.p.m. of