type 1 (HIV-1) regulatory gene, rev (site II or SII), (24). The SII-21 hairpin produces a single siRNA, whereas the SII-50 and SII-80 hairpins are capable of generating two and three individual siRNAs, respectively. These hairpin sequences contain G:U and U:G wobble pairings but do not alter the antisense guide strand of the processed siRNA (Figure 2A). The wobble pairing slightly distorts the stem helix of the hairpin RNA but was shown to facilitate cloning of these long hairpins in E. coli (24). To determine the extent of shRNA or lhRNA processing into 21-mer siRNAs, sh- and lhRNA expression plas- mids were transfected into wild-type and Dicer helicase mutant cells followed by northern blot analyses using 50-end labeled 21-nt oligonucleotides complementary to the antisense guide strand of each siRNA that is predicted to derive from Dicer-processing of the hairpins (Figure 2A and B). Reduced accumulation of siRNAs was observed for the SII-21 shRNA containing G:U wobble base-pair- ing in Dicer helicase mutant cells (Figure 2B), a result that was consistently observed upon repeated transfections, but which did not correlate with the apparent shRNA processing eﬃciency (Figure 2C, Probe 1). Northern ana- lyses of the siRNAs produced from the SII-50 and SII-80 vectors showed somewhat increased amounts of the ﬁrst siRNA processed from the lhRNAs in Dicer helicase mutant cells compared to lhRNA processing in the wild type Dicer background (Figure 2B and C, Probe 1). However, the processing eﬃciency of the second and third siRNAs from these lhRNA vectors was 3–5-fold greater in Dicer helicase mutant cells compared with wild-type cells (Figure 2B and C, Probes 2 and 3). Throughout our experiments we repeatedly observed the inverse accumulation of mature siRNA in wild-type (high- shRNA/low-lhRNA) and Dicer helicase mutant cells (low- shRNA/high-lhRNA) (Figure 2B). Using two diﬀerent luciferase-based reporter systems, we measured a 2-fold increase in the eﬃcacy of target knockdown for the lhRNA vectors in mHelicase cells compared with target knockdown in wild-type cells. In contrast, target knockdown in response to the SII-21 shRNA vector was reduced by about 2-fold in the mHelicase Dicer background, consistent with the reduced steady-state level of processed siRNA determined by northern analysis (Figure 2B and Supplementary Figure 2). The enhanced processing and greater target knockdown in the mHelicase Dicer background is not speciﬁc to the site II HIV lhRNA, as diﬀerent lhRNA vectors targeting the HIV tat/rev common exon (site I or SI), yielded more siRNA and greater eﬃcacy for each lhRNA in the Dicer helicase mutant cells compared to wild-type cells (Supplementary Figure 3). In addition to the increased steady-state levels of the siRNA guide strand from lhRNA vectors in Dicer mHelicase cells, northern analysis revealed stronger sense strand signals for the lhRNA-derived siRNAs in the mHelicase Dicer back- ground relative to wild-type cells (Supplementary Figure 4). Target knockdown by the lhRNA-derived sense strand was also greater in the Dicer mHelicase cells indicating that lhRNA passenger strand siRNAs are also incorporated into RISC (Supplementary Figure 4). No signiﬁcant diﬀerences in sense-strand accumulation or Nucleic Acids Research, 2008, Vol. 36, No. 20 6517 target knockdown from the SII-21 shRNA were observed between wild-type and Dicer mHelicase cells. Taken together, our results suggest that the Dicer mHelicase pro- tein functions markedly more eﬃciently than the wild-type enzyme in the processing of lhRNAs, but not shRNAs containing G:U wobble pairings in the stem. Effects of the Dicer helicase mutation on hairpin RNA processing in vitro To more carefully investigate the role of the hDicer heli- case domain on hairpin RNA processing, we assayed cell extracts prepared from wild-type and Dicer mHelicase cells for their ability to process various hairpin RNA structures in vitro, including a synthetic 60-nt RNA that mimics the pre-miR-21 hairpin RNA, and in vitro tran- scribed RNAs that derive from templates corresponding to the 21-bp shRNA (SII-21) and a 50-bp hairpin (SII-50). Recombinant hDicer was capable of processing all hairpin substrates into 21-nt siRNA products indicating that they are annealed into appropriate hairpin structures (Supplementary Figure 5). Upon incubation with a cell extract prepared from wild-type cells, a 21-nt RNA pro- cessed product was generated from both the pre-miRNA and SII-21 shRNA substrates (Figure 3A and B). Only a small amount of processed siRNA was observed when the SII-50 lhRNA was incubated with the wild-type cell extract (Figure 3B). A 3-fold decrease in the processing eﬃciency of the pre-miR 21 substrate was observed upon incubation with the Dicer mHelicase cell extract, a result consistent with the low levels of endogenous mature miR-21 determined by northern analysis (Figure 3C, Pre- miR21). The processing eﬃciency of the SII-21 shRNA containing G:U wobbles was also reduced by more than 4-fold in the Dicer mHelicase extract (Figure 3C, SII-21). In contrast, the processing eﬃciency of the SII-50 lhRNA was enhanced by more than 6-fold in the extract from the Dicer mHelicase cells relative to SII-50 processing in the wild-type Dicer extract (Figure 3B and C, SII-50). Thermodynamic properties of shRNAs determine the necessity of the Dicer helicase domain To investigate whether structural properties of the shRNA or lhRNA aﬀect processing by hDicer, we constructed SII vectors that express perfectly complementary shRNA (SII-c21) or lhRNAs (SII-c50) by restoring the G:U wobble pairs to their natural base-pairing partners (G:C, A:U) and introduced these vectors into the wild-type and Dicer mHelicase cells for northern blot analysis. As was observed for the G:U wobble-containing lhRNAs, the processing eﬃciency and target knockdown by both the ﬁrst and the second siRNAs generated from the SII-c50 lhRNA was more pronounced in the Dicer mHelicase cells compared with wild-type cells, a result consistent with the increased steady-state levels of mature siRNA in the mHelicase Dicer background (Figure 4 and Supplementary Figure 6). Similarities in the processing eﬃciency of the second siRNA from the perfectly paired lhRNA with lhRNAs containing G:U wobble pairs (compare Figure 2C, probe 2 and Figure 4C, probe 2) were observed suggesting that