The Drosophila Nitric-oxide Synthase Gene (dNOS) Encodes a Family of Proteins That Can Modulate NOS Activity by Acting as Dominant Negative Regulators

Abstract

Nitric oxide (NO) is involved in organ development, synaptogenesis, and response to hypoxia in Drosophila. We cloned and analyzed the only gene in the fly genome that encodes Drosophila nitric-oxide synthase (dNOS). It consists of 19 exons and is dispersed over 34 kilobases of genomic DNA. Alternative transcription start sites and alternative splice sites are used to generate a remarkable variety of mRNAs from the dNOS gene. We identified eight new transcripts that are widely expressed throughout Drosophila development and encode a family of DNOS-related proteins. Alternative splicing affects both the 5-untranslated region and the coding region of the dNOS primary transcript. Most of the splicing alterations in the coding region of the gene lead to premature termination of the open reading frame. As a result, none of the alternative transcripts encode an enzymatically active protein. However, some of these shorter DNOS protein products can effectively inhibit enzymatic activity of the full-length DNOS1 protein when co-expressed in mammalian cells, thus acting as dominant negative regulators of NO synthesis. Using immunoprecipitation, we demonstrate that these short DNOS protein isoforms can form heterodimers with DNOS1, pointing to a physical basis for the dominant negative effect. Our results suggest a novel regulatory function for the family of proteins encoded by the Drosophila NOS gene. Nitric oxide (NO)1 is involved in vasodilation, neurotransmission, and immunity in mammals (see Ref. 1 for review). It is produced by a family of nitric-oxide synthases (NOS), which are encoded by three distinct genes in the mammalian genome. These genes are expressed throughout development as well as in the adult animal. Their transcriptional regulation is highly complex; a range of alternative promoters, splice sites, and polyadenylation signals is used to generate families of transcripts and proteins from each chromosomal gene (see Refs. 2-4 for review).In Drosophila NO has been shown to be involved in imaginal disc development, synaptogenesis, formation of retinal projection pattern, response to hypoxia, and behavioral responses (see Ref. 5 for review). The gene for Drosophila NO synthase, dNOS, is located on the second chromosome at cytological position 32B (6). The major product of the gene, dNOS1, encodes a protein that bears a strong resemblance to all three NOS isoforms of mammals, with the highest homology to neuronal NOS (nNOS) (6). Several attempts to clone putative orthologs of mammalian endothelial or inducible forms of NOS (eNOS or iNOS) from various Drosophila cDNA or genomic libraries have not revealed any other NOS loci in the fruit fly's genome. 2 The recently released complete sequence of the Drosophila genome (7) has further confirmed our conclusion that the dNOS locus represents the only gene for NO synthase in Drosophila.To explore the role of NO in fruit fly physiology and development, we sought to determine the structure of the Drosophila NOS gene to search for alternative transcripts and proteins and...

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“…Table 1. Comparison of the amino acid sequence recognized by the applied rat anti-nNOS antibody and the equivalent NOS amino acid sequences identified in Drosophila (Stasiv, et al, 2001) and some mollusks (Aplysia [Moroz, et al, 2006]; Lymnaea [Korneev et al, 1998[Korneev et al, , 2005). Identical sequences are highlighted.…”

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“…The choice of promoters, splice sites, and polyadenylation sites alters the coding capacity of NOS transcripts as well as their translational efficiency (Wang et al 1999a,b). Likewise, the Drosophila NOS gene (dNOS) encodes a family of at least 10 different transcripts that may code for as many as seven different proteins (Regulski and Tully 1995;Stasiv et al 2001). Of these mRNAs, which are generated through the use of alternative transcription initiation and splice sites, only one isoform, dNOS1, encodes an enzymatically active protein, DNOS1.…”