Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells.
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.
Langlois
Marc-André
MA
Laboratory of Human Genetics, Laval University Medical Research Centre, CHUQ, Pavillon CHUL, Ste-Foy, Quebec G1V 7P4, Canada.
Boniface
Christelle
C
Wang
Gang
G
Alluin
Jessica
J
Salvaterra
Paul M
PM
Puymirat
Jack
J
Rossi
John J
JJ
Lee
Nan Sook
NS
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
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