The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif

Cristiana Griffoni, Pavel P. Laktionov, Elena Y. Rykova, Enzo Spisni, Massimo Riccio, Spartaco Santi, Anton Bryksin, Natalia Volodko, Regine Kraft, Valentin Vlassov, Vittorio Tomasi

Research output: Contribution to journalArticlepeer-review

Abstract

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A2 (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD+ co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.

Original languageEnglish
Pages (from-to)32-46
Number of pages15
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1530
Issue number1
DOIs
Publication statusPublished - Jan 15 2001

Keywords

  • Antisense oligonucleotide
  • Cytosolic phosholipase A
  • Glyceraldehyde-3-phosphate dehydrogenase
  • Nucleus

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Biophysics

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