endogenous pre-miRNAs and the newly discovered oocyte-restricted lhRNAs (32), it is possible that the N-terminal domain of human Dicer evolved to broaden the array of structured hairpin RNAs that this enzyme can eﬃciently cleave. Biochemical and crystallographic data for Dicer and other RNAse III enzymes that lack a heli- case domain have produced models using linear dsRNA molecules that do not take into account the unidirectional entry of Dicer on substrates containing a loop at one end (1,8,33–36). What is clear from these earlier reports, how- ever, is that signiﬁcant conformational changes in the pro- tein and RNA occur during cleavage (8,33,35). Therefore, hairpins with slightly diﬀerent structures due to their sequence composition and thermodynamic stability are likely to require diﬀerent conformational changes in order for processing to be completed. Our own results, as well as those of other labs, have shown a polarity in the processing of long hairpins in cells resulting in reduced amounts and eﬃcacies of distally produced siRNAs (24,37,38). Our current analysis of long hairpin processing in cells harboring either wild-type or mutant helicase Dicer enzymes provide some insight into these previous results and show the Dicer helicase domain restricts the processivity of the enzyme, thereby limiting the eﬀective- ness of long hairpins for intracellular applications in which multiple siRNAs are desired. Steric hindrance of helicase domain function by the inserted residues could provide the mutant helicase Dicer its’ unique sh/lhRNA processing properties. It is possible that the inserted amino acid residues disrupt required con- formational changes that take place after cleavage to bring together the hDicer C-terminal domain, which mediates dsRNA binding and cleavage, with the helicase-containing N-terminus of hDicer. Such an interaction could promote the release of the newly cleaved siRNA duplex. This inter- action between the C- and N-termini of wild-type hDicer is expected to prevent the Dicer C-terminal domain from immediately initiating another round of cleavage resulting in poor processing of lhRNA substrates which require more than one Dicer cleavage event. In the absence of a functional Dicer helicase domain, however, the C-termi- nus of the mutant Dicer enzyme is capable of multiple rounds of hairpin cleavage on long hairpin substrates. In support of this possible mechanism, the recent studies of Ma et al. (30) demonstrate that the major consequence of eliminating the helicase domain from human dicer is a substantial increase in the kcat for cleavage of a perfectly matched 37-nt linear duplex RNA. They suggest that the helicase domain is involved in a rearrangement of the enzyme following catalysis, which restricts the enzyme’s ability to reinitiate cleavage on the long substrates. In the absence of a functional helicase domain this rearran- gement does not occur, thereby allowing the enzyme to rapidly reinitiate cleavage on a long substrate. In contrast to the enhanced cleavage processivity of the mHelicase on long hairpins, short hairpin and pre- miRNA substrates that harbor mismatches in the stem region are cleaved poorly in our system. It is possible that that the complete processing of pre-miRNAs with poor thermodynamic stability properties by Dicer may require interaction with an accessory protein, such as Nucleic Acids Research, 2008, Vol. 36, No. 20 6521 TRBP or PACT. This seems unlikely though since human Dicer can eﬀectively recognize and cleave pre- miRNAs and other hairpin RNA substrates in vitro in the absence of accessory proteins (Supplementary Figure 5). Perhaps a more reasonable explanation for our observations, as well as those of Ma et al. (30), is that once cleavage of an imperfect stem region takes place, the helicase domain helps mediate a conformational rearrangement of the hDicer enzyme to release the guide strand for hand-oﬀ to RISC. In contrast, perfectly matched helices in shRNAs are directly handed oﬀ to Ago 2 wherein the passenger strand is cleaved to create an active RISC (39). Thus, a plausible model for the role of the helicase domain in the human Dicer enzyme is to facilitate product release from substrates with imperfect helices. This may take place via interactions with acces- sory proteins such as TRBP or PACT or through interac- tions between N- and C-terminal domains of the same Dicer molecule. Further structural analyses of the Dicer helicase domain should facilitate a better understanding of why the helicase domain is required for unstable substrate processing and hand-oﬀ but is not required for thermo- dynamically stable duplex processing and handoﬀ. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS The authors would like to thank Bert Vogelstein and Jordan Cummins for the generous gifts of the HCT116 wild-type and Dicer helicase mutant cell lines and the NIH AIDS Research & Reference Reagent Program for providing a pNL4-3.Luc plasmid. Dr. Patrick Provost generously provided the plasmid expressing the wild-type human Dicer cDNA. FUNDING National Institutes of Health (HL07470, AI29329 to J.J.R.); a Beckman Fellowship from the Arnold and Mabel Beckman Foundation (to H.S.S.). Funding for open access charges: NIH grants HL07470 and AI29329 to JJR Conﬂict of interest statement. None declared. REFERENCES 1. Gan,J., Tropea,J.E., Austin,B.P., Court,D.L., Waugh,D.S. and Ji,X. (2006) Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell, 124, 355–366. 2. Gregory,R.I., Yan,K.P., Amuthan,G., Chendrimada,T., Doratotaj,B., Cooch,N. and Shiekhattar,R. (2004) The Microprocessor complex mediates the genesis of microRNAs. Nature, 432, 235–240. 3. MacRae,I.J. and Doudna,J.A. (2007) Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr. Opin. Struct. Biol., 17, 138–145. 4. Carmell,M.A. and Hannon,G.J. (2004) RNase III enzymes and the initiation of gene silencing. Nat. Struct. Mol. Biol., 11, 214–218.