A Novel, Testis-specific mRNA Transcript Encoding an NH2-terminal Truncated Nitric-oxide Synthase

Yang Wang,Michael S Goligorsky,Martin Lin,Josiah N Wilcox,Philip A Marsden

doi:10.1074/jbc.272.17.11392

Yang Wang, Michael S Goligorsky + Show 3 more

Open Access

https://doi.org/10.1074/jbc.272.17.11392

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Abstract

mRNA diversity represents a major theme of neuronal nitric-oxide synthase (nNOS) gene expression in somatic cells/tissues. Given that gonads often express unique and biologically informative variants of complex genes, we determined whether unique variants of nNOS are expressed in the testis. Analysis of cDNA clones isolated from human testis identified a novel, testis-specific nNOS (TnNOS) mRNA transcript. A predicted 3294-base pair open reading frame encodes an NH2-terminal truncated protein of 1098 amino acids. Measurement of calcium-activated L-[14C]citrulline formation and nitric oxide release in CHO-K1 cells stably transfected with the TnNOS cDNA indicates that this protein is a calcium-dependent nitric-oxide synthase with catalytic activity comparable to that of full-length nNOS. TnNOS transcripts exhibit novel 5' mRNA sequences encoded by two unique exons spliced to exon 4 of the full-length nNOS. Characterization of the genomic structure indicates that exonic regions used by the novel TnNOS are expressed from intron 3 of the NOS1 gene. Although lacking canonical TATA and CAAT boxes, the 5'-flanking region of the TnNOS exon 1 contains multiple putative cis-regulatory elements including those implicated in testis-specific gene expression. The downstream promoter of the human nNOS gene, which directs testis-specific expression of a novel NH2-terminal truncated nitric-oxide synthase, represents the first reported example in the NOS gene family of transcriptional diversity producing a variant NOS protein.

Highlights

MRNA diversity represents a major theme of neuronal nitric-oxide synthase gene expression in somatic cells/tissues
Twelve independent clones featured a novel 5Ј-terminus. All of these 12 clones exhibited 155 bp of new sequence upstream of neuronal nitric-oxide synthase (nNOS) exon 4 (Fig. 1). This novel nNOS mRNA transcript, hereafter referred to as testis-specific nNOS (TnNOS), appeared to be expressed at a level comparable to that of the full-length nNOS in the testis as judged by the number of clones isolated in the 5Ј-RACE experiment and assessment of semiquantitative RT-PCR signals
Characterization and analysis of the TnNOS mRNA transcript indicated an open reading frame of 3294 nt, which predicted a protein of 1098 amino acids with a calculated molecular mass of 125,017 Da

Summary

Introduction

MRNA diversity represents a major theme of neuronal nitric-oxide synthase (nNOS) gene expression in somatic cells/tissues. The downstream promoter of the human nNOS gene, which directs testisspecific expression of a novel NH2-terminal truncated nitric-oxide synthase, represents the first reported example in the NOS gene family of transcriptional diversity producing a variant NOS protein. 1 The abbreviations used are: NOS, nitric-oxide synthase(s); nNOS, neuronal NOS; NO, nitric oxide; PIN, protein inhibitor of nNOS; bp, base pair(s); kb, kilobase(s); TnNOS, testis-specific nNOS; nt, nucleotide; RT, reverse transcription; PCR, polymerase chain reaction; UTR, untranslated region; HEPES, N-2-hydroxyethylpiperazine-NЈ-2ethanesulfonic acid; RACE, rapid amplification of cDNA ends. A recent study in 27 families with inherited infantile pyloric stenosis has identified nNOS as a susceptibility gene in this human disorder [28]

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