hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2)

Mol Cell Biochem. 2011 Nov;357(1-2):55-63. doi: 10.1007/s11010-011-0875-5. Epub 2011 May 28.

Abstract

HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.

MeSH terms

  • 3T3 Cells
  • Alternative Splicing / genetics*
  • Animals
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Cell Line, Tumor
  • Exons / genetics
  • Gene Expression
  • HeLa Cells
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H / genetics*
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H / metabolism
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NADH, NADPH Oxidoreductases / genetics*
  • NADH, NADPH Oxidoreductases / metabolism*
  • Neoplasms / enzymology*
  • Protein Isoforms / genetics

Substances

  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H
  • Membrane Proteins
  • Protein Isoforms
  • NADH, NADPH Oxidoreductases
  • tumor-associated NADH oxidase