title: Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells.
abstract: 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 ( approximately 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.
authors.affiliation: Laboratory of Human Genetics, Laval University Medical Research Centre, CHUQ, Pavillon CHUL, Ste-Foy, Quebec G1V 7P4, Canada.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Langlois
authors.forename: Christelle
authors.initials: C
authors.lastname: Boniface
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Jessica
authors.initials: J
authors.lastname: Alluin
authors.forename: Paul M
authors.initials: PM
authors.lastname: Salvaterra
authors.forename: Jack
authors.initials: J
authors.lastname: Puymirat
authors.forename: John J
authors.initials: JJ
authors.lastname: Rossi
authors.forename: Nan Sook
authors.initials: NS
authors.lastname: Lee
mesh_terms: D021581:Active Transport, Cell Nucleus; D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D015536:Down-Regulation; D006579:Heterozygote; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology; D009223:Myotonic Dystrophy / Q000378:metabolism*; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013347:Subcellular Fractions; D014162:Transfection
chemical_list: C585517:DMPK protein, human; D016337:RNA, Catalytic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C418481:hairpin ribozyme; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases