Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes

Nucleic Acids Res. 2003 Sep 1;31(17):4973-80. doi: 10.1093/nar/gkg707.

Abstract

Micro-RNAs are a class of small non-coding regulatory RNAs that impair translation by imperfect base pairing to mRNAs. For analysis of their cellular function we injected different miRNA-specific DNA antisense oligonucleotides in Drosophila embryos. In four cases we observed severe interference with normal development, one had a moderate impact and six oligonucleotides did not cause detectable phenotypes. We further used the miR-13a DNA antisense oligonucleotide as a PCR primer on a cDNA library template. In this experimental way we identified nine Drosophila genes, which are characterised by 3' untranslated region motifs that allow imperfect duplex formation with miR-13 or related miRNAs. These genes, which include Sos and Myd88, represent putative targets for miRNA regulation. Mutagenesis of the target motif of two genes followed by transfection in Drosophila Schneider 2 (S2) cells and subsequent reporter gene analysis confirmed the hypothesis that the binding potential of miR-13 is inversely correlated with gene expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Cell Line
  • DNA, Antisense / administration & dosage
  • DNA, Antisense / genetics*
  • Drosophila / cytology
  • Drosophila / embryology
  • Drosophila / genetics*
  • Embryo, Nonmammalian / abnormalities
  • Embryo, Nonmammalian / drug effects
  • Embryo, Nonmammalian / metabolism
  • Gene Expression Regulation, Developmental
  • Genes, Insect / genetics*
  • Luciferases / genetics
  • Luciferases / metabolism
  • MicroRNAs / genetics*
  • Mutation
  • Oligonucleotides, Antisense / administration & dosage
  • Oligonucleotides, Antisense / genetics
  • Protein Biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transfection

Substances

  • DNA, Antisense
  • MicroRNAs
  • Oligonucleotides, Antisense
  • Recombinant Fusion Proteins
  • Luciferases