title: 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.affiliation: NOX Technologies, Inc, Purdue University Research Park, West Lafayette, IN 47906, USA.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Tang
authors.forename: Vanessa D
authors.initials: VD
authors.lastname: Kane
authors.forename: Dorothy M
authors.initials: DM
authors.lastname: Morré
authors.forename: D James
authors.initials: DJ
authors.lastname: Morré
mesh_terms: D016475:3T3 Cells; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000235:genetics* / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D009369:Neoplasms / Q000201:enzymology*; D020033:Protein Isoforms / Q000235:genetics
chemical_list: D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D008565:Membrane Proteins; D020033:Protein Isoforms; D009247:NADH, NADPH Oxidoreductases; C431909:tumor-associated NADH oxidase