Abstract
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.
Highlights
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
NOH-1S was not detected by Northern blotting of colon cells, a site of high Nox1 expression, despite the suggestion of its relative abundance based on RNase protection assays [18]
The deleted NOH-1S could only be generated from colon cell RNA when using avian myeloblastosis virus (AMV) reverse transcriptase at low temperatures, whereas only the large Nox1 transcripts were synthesized when using a reverse transcriptase (ThermoScriptTM) that performs at higher temperatures
Summary
Vol 279, No 49, Issue of December 3, pp. 51661–51668, 2004 Printed in U.S.A. EVIDENCE AGAINST PRODUCTION OF THE NADPH OXIDASE HOMOLOG-1 SHORT (NOH-1S) TRANSCRIPT VARIANT*. We characterized Nox 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 notion that gp91phox functions as a proton channel was supported further by studies on the gp91phox homolog Nox, where a putative alternatively spliced product The putative product, which was not detected at the protein level, consists of four transmembrane domains and a short cytoplasmic tail; the recombinant protein, when expressed ectopically in HEK 293 cells, exhibited the properties of voltage-gated Hϩ channels We show that the mRNA transcript for NOH-1S is not synthesized from the Nox gene but is instead an artifactual product of cDNA synthesis We attribute this result to the phenomenon of intramolecular template switching between repetitive sequence elements during cDNA synthesis. We demonstrate that the Nox gene encodes another genuine spliced variant of Nox that is abundant in colon cells but is not functional because of the absence of exon 11-encoded sequence
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