in the presence of 32P-a-UTP according to the manufac- turer’s instructions and gel puriﬁed to obtain the full- length RNA. 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 reac- tions using rDicer, 5 104 c.p.m. of labeled sh- or lhRNA was incubated with 1 U of rDicer (Genlantis) according the manufacturer’s instructions. For in vitro reactions using cell extracts from wild-type or Dicer mHelicase cells, 5 104—1 105 c.p.m.of labeled sh- or lhRNA was incubated with 25 mg of cell extract, 3.2 mM MgCl2 and 0.5 mM ATP at 378C for 1–3 h. Reactions were stopped by the addition of EDTA and immediately run in 10–15%/8 M Urea denaturing polyacrylamide gels. Following electrophoresis, separated RNAs were analyzed by exposing the gel to a phosphorscreen and analyzed using a Storm phosphorimager. The processing eﬃciency for each hairpin RNA was determined as follows: proces- sing eﬃciency = matureIDV/(matureIDV + precursorIDV) IDV, Integrated Density Value. RESULTS Characterization of the Dicer helicase mutant HCT116 cells A frozen aliquot of a subclone of HCT116 cells that con- tains a targeted insertion in exon 5 of both alleles of Dicer, herein referred to as the Dicer helicase mutant (or mHelicase) cells, along with the parental wild-type HCT116 cells, were kindly provided by Dr Bert Vogelstein (23). PCR ampliﬁcation and sequence analysis of genomic DNA from wild-type and Dicer helicase mutant cells using primers that ﬂank exon 5 of the human Dicer gene indicate that the Dicer helicase mutant cells contain a 129-bp insertion of an ectopic sequence derived from the targeting vector that is in- frame with the predicted amino acid sequence of Dicer (Supplementary Figure 1A). The insertion mutation resides two amino acids from the DExH motif DECH, an important motif in RNA helicases (29). Whereas the insertion mutation had no apparent eﬀect on the level of hDicer mRNA in Dicer mHelicase cells (Supplementary Figure 1C), the level of hDicer protein is reduced in Dicer mHelicase cells compared with wild-type HCT116 cells (Supplementary Figure 1D). Because of the absence of structural data for the hDicer N-terminus, we cannot accurately determine the structural perturbations in the helicase induced by the 43-amino-acid insertion, nor can we rule out the possibility that other Dicer domains are aﬀected by the insertion mutation. Based on empirical evidence presented below, showing the eﬃcient processing of some endogenous pre-miRNAs and ectopic shRNAs in the mHelicase cells, we anticipate that the insertion into the helicase domain does not aﬀect other functional domains of hDicer, such as the PAZ and C-terminal cleav- age domains. By homology modeling, using the crystal structure of the Hef helicase from P. furiosus (26) that has sequence similarity with the Dicer helicase domain, we predict that the 43-amino-acid insertion disrupts the Hef DECH box and other motifs involved in ATP Nucleic Acids Research, 2008, Vol. 36, No. 20 6515 hydrolysis by the Figure 1E) (26,29). Hef helicase (Supplementary Processing and efficacy of miRNAs in wild-type and the Dicer helicase mutant cells While the expression of many miRNAs is reduced in mHelicase cells, as shown by the marked reduction in mature miR-21 (Figure 1A), a closer examination of the miRNA expression data generated by Cummins et al. (23) indicate that 25% of the 100 most highly expressed miRNAs in the wild-type and mHelicase cells did not exhibit signiﬁcant changes in miRNA expression (data not shown). Our own northern blot analyses for two of these unaﬀected miRNAs (miR-23a and miR-27a) conﬁrm that the processing and expression of these miRNA is the same in both wild-type and mHelicase cells (Figure 1A). In addition, transient expression of ectopic perfectly comple- mentary shRNA (SII-c21) or lhRNA (SII-c50) targeting the human immunodeﬁciency virus type 1 (HIV-1) regu- latory gene, rev (site II or SII) in wild-type and mHelicase cells had no eﬀect on the expression of miR-23a and miR- 27a (Figure 1A). The observation that multiple miRNAs escape downregulation in the mHelicase background sug- gests that the reduced level of Dicer is not limiting for pre- miRNA cleavage in these cells. Moreover, the fact that the expression of miR-23a and miR-27a remains unchanged in both cell lines that express ectopic shRNA or lhRNA indicates that transient transfection does not have a major impact on miRNA expression or the viability of the mHelicase cells. The expression of miR-21, whose steady-state level is reduced in mHelicase cells, also shows reduced expression following transient transfection of diﬀerent shRNA or lhRNA vectors (SII-21 and SII-50; Figure 2). To determine whether the reduced production of some miRNAs in mutant cells has a detrimental eﬀect on miRNA target knockdown, we cloned a polycistronic miRNA (miR-106b-93-25) from the MCM7 gene down- stream of the CMV promoter and constructed a separate reporter vector that contains a target site perfectly com- plementary to miR-93 in the 30 UTR of the Renilla luci- ferase gene (25). All three miRNAs that originate from this MCM7 cluster are reduced in the Dicer mHelicase cells (23). In wild-type cells, the MCM7 vector reduced miR-93 target gene expression by almost 70% compared with an irrelevant control vector (Figure 1B). In contrast, the eﬃcacy of target knockdown was reduced 2-fold in the Dicer helicase mutant cells (Figure 1B). Upon co-transfection of Dicer helicase mutant cells with a plas- mid expressing wild-type Dicer cDNA (7), target knock- down was restored to near wild-type levels, showing that the mutation is not dominant (Figure 1B). Processing and efficacy of the shRNA or lhRNA in wild-type and the Dicer helicase mutant cells To gain additional insight into the role of the hDicer heli- case domain in hairpin processing, we used previously reported plasmids which express a 21-bp shRNA (SII- 21), 50-bp lhRNA (SII-50) or 80-bp lhRNA (SII-80) targeted against the human immunodeﬁciency virus