Published online 15 October 2008 Nucleic Acids Research, 2008, Vol. 36, No. 20 6511–6522 doi:10.1093/nar/gkn687 A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs Harris S. Soifer1, Masayuki Sano 2 , Kumi Sakurai1,3, Pritsana Chomchan 1 , Pa˚ l Sætrom4,7, Mark A. Sherman5, Michael A. Collingwood6, Mark A. Behlke6 and John J. Rossi1,3,* 1Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA, 2Biotherapeutic Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan, 3Graduate School of Biological Sciences, City of Hope, Duarte, CA 91010, USA, 4Department of Computer and Information Science, Norwegian University of Science and Technology, Sem Sælands vei 7-9, NO-7491 Trondheim, Norway, 5Department of Biomedical Informatics, Division of Information Sciences, City of Hope, Duarte, CA 91010, USA, 6Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA and 7Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology and Interagon AS, Laboratoriesenteret, NO-7489 Trondheim, Norway Received August 28, 2008; Revised September 22, 2008; Accepted September 24, 2008 ABSTRACT In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that pos- sesses a signature DExH motif. Cummins et al. cre- ated a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in- frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and compu- tational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermody- namic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically uns- table stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced pro- cessing efficiency and concomitant RNA interfer- ence when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the proces- sing of thermodynamically unstable hairpin structures. INTRODUCTION Eukaryotic ribonuclease III (RNase III) enzymes exhibit speciﬁcity for double-stranded RNA (dsRNA) and are divided into three structural classes. The ﬁrst class of RNase III enzymes are the simplest, consisting of a single catalytic domain (RIII), and are represented by Escherichia coli RNase III that promotes ribosomal RNA (rRNA), tRNA and mRNA maturation through cleavage of hairpin-like stem–loop structures (1). Members of the second RNase III class, which include Drosha and its homologs, contain two catalytic RIII domains, a dsRNA binding domain (dsRBD), and an N-terminal region that mediates interactions with protein partners such as DGCR8 (2). The third class of RNase III enzymes comprised of Dicer and Dicer-like proteins that play essential roles in several developmental processes through the processing of endogenous dsRNA substrates. For the most part, Dicer enzymes contain an N-terminal DExH-box RNA helicase-like domain, a domain of unknown function (DUF283), a PAZ domain, two RIII domains (RIIIa and RIIIb), and a dsRBD (3). The PAZ domain is highly conserved and is also found in proteins *To whom correspondence should be addressed. Tel: +1 626 301 8360; Fax: +1 626 301 8271; Email: jrossi@coh.org. The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.