Nucleic Acids Research Advance Access originally published online on October 15, 2008
Nucleic Acids Research 2008 36(20):6511-6522; doi:10.1093/nar/gkn687
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Nucleic Acids Research, 2008, Vol. 36, No. 20 6511-6522
© 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.
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A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs
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
*To whom correspondence should be addressed. Tel: +1 626 301 8360; Fax: +1 626 301 8271; Email: jrossi{at}coh.org.
Received August 28, 2008. Revised September 22, 2008. Accepted September 24, 2008.
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 possesses a signature DExH motif. Cummins et al. created 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 computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors