An Alternatively Spliced Form of NQO1 (DT-Diaphorase) Messenger RNA Lacking the Putative Quinone Substrate Binding Site Is Present in Human Normal and Tumor Tissues

DT-diaphorase is a ubiquitously expressed flavoenzyme responsible for the two-electron reduction of a number of quinone and other anticancer drugs. The majority of DT-diaphorase enzyme activity in human tissues is the product of the NQO₁ gene. We have now identified a novel alternatively spliced form of human NQO₁, mRNA lacking exon 4 at levels equal to or exceeding those of wild-type NQO₁, mRNA. Exon 4 codes for the putative quinone substrate binding site of DT-diaphorase derived from NQO₁, and the recombinant protein from alternatively spliced NQO₁, mRNA lacking exon 4 has minimal enzyme activity with quinoid and other known substrates of DT-diaphorase. The physiological substrate of DT-diaphorase is unknown, and it is possible that the protein derived from the alternatively spliced NQO₁, mRNA could have enzyme activity with an appropriate substrate. We found full-length DT-diaphorase protein but could not detect expression of an appropriately smaller form of DT-diaphorase in human tissues using polyclonal antibody to DT-diaphorase, suggesting that alternatively spliced NQO₁, mRNA lacking exon 4 may not be translated or that the protein product is rapidly degraded. Alternative splicing of NQO₁, RNA could provide an important mechanism for regulating NQO₁ gene expression.