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Nucleic Acids Research, 2003, Vol. 31, No. 17 4973-4980
© 2003 Oxford University Press

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

Alexandra Boutla1,2, Christos Delidakis1,2 and Martin Tabler*,1

1 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Hellas, PO Box 1527, GR-71110 Heraklion/Crete, Greece and 2 Department of Biology, University of Crete, GR-71110 Heraklion/Crete, Greece

*To whom correspondence should be addressed. Tel: +30 2810 394 365; Fax: +30 2810 394 408; Email: tabler{at}imbb.forth.gr

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.


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