6520 Nucleic Acids Research, 2008, Vol. 36, No. 20 Figure 5. Structural properties of short hairpin precursors aﬀect their processing in Dicer mHelicase cells. (A) Northern blot detection of siRNAs derived from various shRNAs in the wild type or mHelicase cells. Northern blotting analyses were performed as described in Figure 3. The mature siRNA is indicated with the arrowhead and the precursor molecules are indicated by the vertical bar. The membrane was subsequently stripped and probed for miR-21. U6 snRNA expres- sion was also detected as a loading standard. The illustrated size marker is based on the migration of the Ambion Decade ladder. (B) The processing eﬃciency for each shRNA was determined by autora- diography and dosimetry as follows: processing eﬃciency = matureIDV/ (matureIDV + precursorIDV). IDV, Integrated Density Value. The mean and standard deviation of three experiments is represented. at the end of the stem (positions 1–3) or around the Dicer processing site (positions 18–22) were cloned less fre- quently in the Dicer helicase mutant cells compared with wild-type cells (Supplementary Figure 7 and Figure 1A) (23). In light of these observations, we sought to carry out a systematic approach to delineate the role of the hDicer helicase domain in processing shRNAs. To do this, we constructed several additional shRNA variants by insert- ing G:U wobble pairings or creating bulges within the stem of the Sh-c21 shRNA vector and performed northern blot analysis for processed siRNA. Since each shRNA contains diﬀerent G:U wobble pairs and/or bulges within the stem, the resultant shRNAs have diﬀerent cal- culated thermodynamic stabilities (Supplementary Figure 8A). In both cell lines, the processing of SII-21 shRNA variants with lower thermodynamic stabilities was reduced compared with the parental SII-c21 vector (Figure 5). In particular, the processing eﬃciency of SII- G26, which has a similar predicted thermodynamic stabi- lity to the pre-miR-21 hairpin ( G = –24.9 kcal/mol), was most aﬀected by the Dicer helicase mutation. The steady- state ratios of the endogenous pre-miR21/mature miR-21 are also increased in the Dicer mHelicase cells (Figure 5A). Even though the processing eﬃciency of most SII-21 shRNA variants did not diﬀer signiﬁcantly between wild-type and Dicer mHelicase cells (Figure 5B), the eﬃ- cacy of target knockdown was signiﬁcantly reduced in the mHelicase Dicer background for most shRNAs with lower internal stabilities (Supplementary Figure 8B), consistent with the impaired production of siRNAs and suggestive of a defect in guide strand handoﬀ to RISC by the Dicer mHelicase mutant. DISCUSSION Human Dicer possesses several domains that are required for functional processing of pre-miRNAs as well as longer double-stranded RNAs. Prior to this study and a recent publication by Ma et al. (30) the role of the N-terminal helicase domain in Dicer function was unstudied. We have taken advantage of a human cell line that expresses a mutant form of hDicer that has an in-frame, 43-amino- acid insertion immediately adjacent to the DExH motif in the N-terminal helicase domain. This mutant Dicer enzyme exhibits poor processing of pre-miRNAs and shRNA with thermodynamically unstable stem regions, particularly when normal Watson–Crick base pair inter- actions are disrupted at the base of the stem (positions 1–3) and close to the Dicer cleavage site (i.e. base-paired between nucleotides 18–22) (Figure 1A and Supplemen- tary Figure 7). By restoring Watson–Crick base pairing in the stem region of an shRNA, the processing eﬃciency and target knockdown in the mHelicase Dicer background could be restored to levels achieved in wild-type cells. A somewhat surprising characteristic of the Dicer mHelicase is the enhanced processing of lhRNA with stem regions 50-nt. The processing of endogenous lhRNAs by Dicer in mammals has gained importance with the discovery of long RNAs in mouse oocytes that appear to regulate gene expression through Dicer- and Ago 2-dependent pro- cesses (31,32). Taken together, our results provide strong evidence that the helicase domain is required for eﬃcient processing and possibly handoﬀ of the guide strand to RISC when the substrates are thermodynamically unstable duplexes. Eukaryotes possessing only one Dicer gene that lack the helicase domain, such as Giardia and Schizosaccharomyces Pombe, encounter only perfectly matched duplex or hair- pin RNAs and do not encode known miRNAs (31). Given the varied sequence and secondary structures of human