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.
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Abbreviations
- ECTO-NOX:
-
Cell-surface and growth-related NADH oxidase with protein disulfide-thiol interchange activity
- ENOX2 (tNOX):
-
Tumor-associated and drug-responsive ECTO-NOX of cancer cells
- HeLa cells:
-
Human cervical carcinoma cell line
- HUVEC:
-
Human umbilical vein endothelial cells
- ESS:
-
Exonic splicing silencer
References
Morre DJ, Pogue R, Morré DM (1999) A multifunctional ubiquinol oxidase of the external cell surface and sera. BioFactors 9:179–187
Morré DJ, Morré DM (2003) Cell surface NADH oxidases (ECTO-NOX proteins) with roles in cancer cellular time-keeping, growth, aging and neurodegenerative diseases. Free Radic Res 37:795–808
Kelker M, Kim C, Chueh PJ, Guimont R, Morré DM, Morré DJ (2001) Cancer isoform of a tumor-associated cell surface NADH oxidase (tNOX) has properties of a prion. Biochemistry 40:7351–7354
Morré DJ, Reust T (1997) A circulating form of NADH oxidase activity responsive to the antitumor sulfonylure N-4-(methylphenylsulfonyl)-N′-(4-chlorophenyl) urea (LY181984) specific to sera from cancer patients. J Bioenerg Biomembr 29:281–289
Morré DJ, Chueh PJ, Morré DM (1995) Capsaicin inhibits preferentially the NADH oxidase and growth of transformed cells in culture. Proc Natl Acad Sci USA 92:1831–1835
Morré DJ, Caldwell S, Mayorga A, Wu L, Morré DM (1997) NADH oxidase activity from sera altered by capsaicin is widely distributed among cancer patients. Arch Biochem Biophys 342:224–230
Chueh P-J, Wu L, Morré DM, Morré DJ (2004) tNOX is both necessary and sufficient as a cellular target for the anticancer actions of capsaicin and the green tea catechin (-)-epigallocatechin-3-gallate. Bio Factors 20:235–249
Morré DJ, Bridge A, Wu L, Morré DM (2000) Preferential inhibition by (-)-epigallocatechin-3-gallate of the cell surface NADH oxidase and growth of transformed cells in culture. Biochem Pharmacol 60:937–946
Tang X, Tian Z, Chueh PJ, Chen S, Morré DM, Morré DJ (2007) Alternative splicing as the basis for specific localization of tNOX, a unique hydroquinone (NADH) oxidase, to the cancer cell surface. Biochemistry 46:12337–12346
Yagiz K, Morré DJ, Morré DM (2006) Transgenic mouse line overexpressing the cancer-specific tNOX protein has an enhanced growth and acquired drug-response phenotype. J Nutr Biochem 17:750–759
Cho NM, Morré DJ (2008) Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells. Cancer Immunol Immunother 58:547–552
Cho NM, Chueh P-J, Kim C, Caldwell S, Morré DM, Morré DJ (2002) Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients. Cancer Immunol Immunother 51:121–129
Chang LY, Ali AR, Hassan SS, AbuBakar S (2007) Human neuronal cell protein responses to Nipah virus infection. Virol J 4:54
Wang Z, Rolish M, Yeo G, Tung V, Mawson M, Burge C (2004) Systematic identification and analysis of exonic splicing silencers. Cell 119:831–845
Caputi M, Zahler A (2001) Determination of the RNA binding specificity of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/H’/F/2H9 family. J Biol Chem 276:43850–43859
Matunis M, Xing J, Dreyfuss G (1994) The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization. Nucl Acids Res 22:1059–1067
Honoré B, Rasmussen HH, Vorum H, Dejgaard K, Liu X, Gromov P, Madsen P, Gesser B, Tommerup N, Celis JE (1995) Heterogeneous nuclear ribonucleoproteins H, H′, and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes. J Biol Chem 270:28780–28789
Barberan-Soler S, Zahler AM (2008) Alternative splicing regulation during C. elegans development: splicing factors as regulated targets. PLoS Genet 4:e1000001
Hai Y, Cao W, Liu G, Hong SP, Elela SA, Klinck R, Chu J, Xie J (2008) A G-tract element in apoptotic agents-induced alternative splicing. Nucl Acids Res 36:3320–3331
Honoré B, Baandrup U, Vorum H (2004) Heterogeneous nuclear ribonucleoproteins F and H/H′ show differential expression in normal and selected cancer tissues. Exp Cell Res 294:199–209
Grellscheid S, Smith C (2006) An apparent pseudo-exon acts both as an alternative exon that leads to nonsense-mediated decay and as a zero-length exon. Mol Cell Biol 26:2237–2246
Vela E, Roca X, Isamat M (2006) Identification of novel splice variants of the human CD44 gene. Biochem Biophys Res Commun 343:167–170
Karni R, de Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR (2007) The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol 14:185–193
Acknowledgments
We thank Charles Kuntz for conduct of the experiments of Fig. 1, Braxton Stewart for preliminary studies to identify the splicing factor directing formation of the ENOX2 splice variant, and Peggy Runck for assistance in manuscript preparation.
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Tang, X., Kane, V.D., Morré, D.M. et al. hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2). Mol Cell Biochem 357, 55–63 (2011). https://doi.org/10.1007/s11010-011-0875-5
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DOI: https://doi.org/10.1007/s11010-011-0875-5