Analysis of mRNA transcripts from the NAD(P)H oxidase 1 (Nox1) gene. Evidence against production of the NADPH oxidase homolog-1 short (NOH-1S) transcript variant.
Recent reports indicate that NAD(P)H oxidase 1 (Nox1) mRNA undergoes alternative splicing, producing a short transcript (NOH-1S) encoding a novel H+ channel. Although the H+ transport properties of NOH-1S-transfected cells resemble those of many cells, the production of a NOH-1S protein was never documented. We characterized Nox1 transcripts in colon-derived cells and present evidence that mRNA splicing does not produce NOH-1S; rather, NOH-1S appears to be an artifact of template switching during cDNA synthesis. The NOH-1S transcript was not observed by Northern blotting, despite claims of its abundance based on RNase protection assays. The shortened cDNA was generated by avian myeloblastosis virus reverse transcriptase, but not by thermally stable reverse transcriptase under conditions that produce full-length Nox1. Analysis of shortened cDNAs detected NOH-1S sequence and other variants that differ at the alleged splice junction site. Although no appropriate RNA splicing sites were found within Nox1 to account for NOH-1S formation, we found repetitive sequence elements bordering the deleted region, which could promote intramolecular template switching during cDNA synthesis. Template switching was confirmed in vitro, where the deleted cDNA was generated by avian myeloblastosis virus reverse transcriptase from a synthetic, full-length Nox1 RNA template. A survey of the expressed sequence tags database suggests that similar switching phenomena occur between repetitive elements in other Nox family transcripts, indicating such cloning artifacts are common. In contrast, genuine RNA splicing does account for another Nox1 transcript lacking the entire exon 11, which is abundant in colon cells but encodes a protein incapable of supporting superoxide production.
Geiszt
Miklós
M
Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
Lekstrom
Kristen
K
Leto
Thomas L
TL
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D003106:Colon / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D005091:Exons; D014644:Genetic Variation; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D032446:Myoblasts / Q000378:metabolism; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011522:Protons; D012333:RNA, Messenger / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000737:chemistry; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; D011522:Protons; D012333:RNA, Messenger; D013481:Superoxides; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D012260:Ribonucleases