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

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.

• Authors: Xiaoyu Tang, Vanessa D Kane, Dorothy M Morré, D James Morré
• Journal: Molecular and cellular biochemistry (Mol Cell Biochem) Vol. 357 Issue 1-2 Pg. 55-63 (Nov 2011) ISSN: 1573-4919 [Electronic] Netherlands
• PMID: 21625959 (Publication Type: Journal Article)

Chemical References

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

Topics

• 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, metabolism)
• Human Umbilical Vein Endothelial Cells
• Humans
• Membrane Proteins (genetics, metabolism)
• Mice
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• NADH, NADPH Oxidoreductases (genetics, metabolism)
• Neoplasms (enzymology)
• Protein Isoforms (genetics)