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J. Biol. Chem., Vol. 280, Issue 17, 16949-16954, April 29, 2005

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Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells^*

Marc-André Langlois, Christelle Boniface, Gang Wang¶, Jessica Alluin¶, Paul M. Salvaterra||, Jack Puymirat, John J. Rossi¶, and Nan Sook Lee¶**

From the Laboratory of Human Genetics, Laval University Medical Research Centre, CHUQ, Pavillon CHUL, Ste-Foy, Quebec G1V 7P4, Canada and the ^¶Division of Molecular Biology and ^||Division of Neuroscience, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats (3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.

Received for publication, February 10, 2005

^* This work was funded in part by a grant from the Muscular Dystrophy Association (MDA) (to N. S. L.) and by grants from the MDA (to J. P.), the Canadian Institutes of Health Research (to J. P. and J. J. R.), the Muscular Dystrophy Association of Canada (MDAC) (to J. P.), and from the l'Association Française contre les myopathies (AFM) and the National Institutes of Health (to J. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Doctoral Studentship Award from the Canadian Institutes of Health Research. Present address: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom.

^** To whom correspondence should be addressed. Tel.: 626-301-8296; Fax: 626-301-8072; E-mail: nlee{at}coh.org.

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