6512 Nucleic Acids Research, 2008, Vol. 36, No. 20 from the Argonaute superfamily that are important med- iators of RNA interference (4). Mammalian genomes harbor one Dicer gene encoding a 220-KDa protein that has the multidomain structure typical of class 3 RNase III enzymes (5,6). In vitro experi- ments using wild-type and various mutant versions of the human Dicer enzyme (hDicer) reveal that hDicer can pro- cess various forms of dsRNA, including long linear dsRNA molecules that contain 50 PO4 and 30 OH groups, as well as short hairpin RNAs (shRNA) that mimic Drosha-produced pre-miRNAs (5,7,8). For long dsRNA, cleavage by hDicer into 21-mer siRNAs can occur from both ends of the open, linear substrate in an ATP-independent manner (7–10). In contrast, processing of pre-miRNAs into mature miRNAs occurs through recognition of the 2-nt 30 overhang at one end of the hair- pin duplex (11). Analysis of dsRNA processing by hDicer enzymes engineered with speciﬁc RIII domain mutations reveal that human Dicer contains a single dsRNA proces- sing center formed through intramolecular dimerization of the two RIII domains that function independently to cleave phosphodiester bonds on opposite strands of the dsRNA substrate (12,12). Biochemical analysis of hDicer, along with crystal structure data of the hDicer C-terminus, indicates that divalent cations (e.g. Mg2+) are present at the active site and are required for catalysis (7,12,13). At present, the crystal structure of the hDicer N-terminus has not been reported. Biochemical fractionation of human cells indicates that hDicer does not exist alone, but rather associates in vivo with other dsRNA-binding partners such as members of the Ago family (14,15), HIV-1 TAR RNA-binding protein (TRBP) (16,17) and a related protein, protein activator of PKR (PACT) (18,19), RNA helicase A (RHA) (20) and possibly others. RNAi depletion of PACT or TRBP has a detrimental eﬀect on miRNA-mediated and siRNA- mediated gene silencing, respectively, with loss of PACT expression resulting in a decrease in mature miRNA levels (19). Dicer is also found in a complex with Ago 2, the slicer component of RISC (21), suggesting that Dicer is involved in the hand-oﬀ of processed trigger molecules for use by RISC (17,22). Therefore, the processing of hairpin RNAs by hDicer is likely to occur in multiple steps that involve diﬀerent domains of the enzyme. The contribu- tions of other proteins such as Ago 2 and TRBP, which have been identiﬁed in a putative miRNA loading com- plex are likely to also be important for eﬃcient hairpin processing (14,15). Despite biochemical and crystallographic investigation of the hDicer C-terminal domain, the role of the N-terminal helicase domain in hDicer activity is still poorly understood. Recently a Dicer mutant cell line was created via AAV insertional mutagenesis of the hDicer helicase domain (23). This cell line has reduced levels of many, but not all, miRNAs relative to its wild- type parental cell line. We have further characterized the nature of the insertion, which is a homozygous in-frame 43-amino-acid insertion close to the helicase DExH motif. Our ﬁndings demonstrate that this mutation has detrimen- tal eﬀects on the processing of most pre-miRNAs, but paradoxically has enhanced processing relative to wild-type hDicer of long-hairpin transcripts. These studies suggest an important role for the helicase domain in the reco- gnition and processing of imperfect Watson–Crick helices, and the absence of a functional helicase domain results in enhanced processivity in the cleavage of long hairpins. Our studies lead to a model in which the hDicer helicase domain is required for eﬃcient processing of hairpin RNAs with thermodynamically unstable stem regions. Most likely this characteristic of hDicer reﬂects upon the requirement that Dicer processes a variety of stem–loop substrates, pointing to an important role for the helicase domain in expanding the function of human Dicer beyond pre-miRNA processing. Interestingly, perturbation of this domain results in enhanced processivity by the enzyme on long, thermodynamically stable hairpin RNAs. MATERIALS AND METHODS Plasmid construction The plasmids expressing sh- and lhRNA that target the HIV genome are driven by the human U6 promoter and are described in detail elsewhere (24). Luciferase reporter plasmids were constructed by inserting the annealed oli- gonucleotides that contain the relevant target site were inserted into XhoI and NotI sites of the psiCheck-2 plasmid (Promega) and were previously described in detail (24,25). The plasmid expressing wild-type Dicer was a generous gift of Dr. Patrick Provost (7). Cell culture Wild-type HCT116 and Dicer mHelicase cells, an HCT116-based cell line that contains a targeted insertion into exon 5 of both Dicer alleles was generously provided by Dr Bert Vogelstein (23). Cells were cultured in Dulbecco’s modiﬁed Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 20 U/ml penicillin/strepto- mycin and 2 mM L-Gln. Cells were grown in a humidiﬁed, 5% CO2 incubator at 378C and were passaged by standard means. Mutant Dicer characterization Genomic DNA was collected from wild-type or mHelicase cells by standard methods and subjected to PCR with primers DSF (50 GTTCTCATTATGACTTGCTATGTC GC) and DSR (50 CTCGATAGGGGTGGTCTAGGA TT) under the following cycling conditions: 958C, 5 min, one cycle; 948C, 30 s, 558C, 30 s, 728C, 30 s, 30 cycles; 728C, 10 min, one cycle. PCR products were examined by electrophoresis and cloned into a T/A cloning vector followed by sequencing with vector-speciﬁc primers. The in-frame mutation in the helicase domain was identiﬁed and used in BLAST queries to determine its origin. For quantitative RT-PCR, total RNA from wild-type and Dicer mHelicase cells was collected with RNA STAT- 60 (Tel-Test) and converted into cDNA using random hexamers and M-MLV reverse transcriptase. Control reactions omitting M-MLV were also included to rule- out genomic DNA contamination. Prepared cDNA was subjected to separate real-time PCR reactions using the iQ