Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells

J Biol Chem. 2005 Apr 29;280(17):16949-54. doi: 10.1074/jbc.M501591200. Epub 2005 Feb 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Active Transport, Cell Nucleus
  • Alleles
  • Base Sequence
  • Binding Sites
  • Blotting, Northern
  • Blotting, Western
  • Cell Differentiation
  • Cell Line
  • Cell Nucleus / metabolism*
  • Cloning, Molecular
  • Cytoplasm / metabolism*
  • Down-Regulation
  • Heterozygote
  • Humans
  • Lentivirus / genetics
  • Microscopy, Fluorescence
  • Models, Genetic
  • Molecular Sequence Data
  • Muscle, Skeletal / cytology
  • Myotonic Dystrophy / metabolism*
  • Myotonin-Protein Kinase
  • Protein Serine-Threonine Kinases / metabolism*
  • RNA Interference
  • RNA Processing, Post-Transcriptional
  • RNA, Catalytic / chemistry
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism
  • Subcellular Fractions
  • Transfection

Substances

  • DMPK protein, human
  • RNA, Catalytic
  • RNA, Messenger
  • RNA, Small Interfering
  • hairpin ribozyme
  • Myotonin-Protein Kinase
  • Protein Serine-Threonine Kinases