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

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 NH 2-terminal truncated protein of 1098 amino acids. Measurement of calcium-activated L-[ 14 C]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 testisspecific expression of a novel NH 2-terminal truncated nitric-oxide synthase, represents the first reported example in the NOS gene family of transcriptional diversity producing a variant NOS protein.

An Alternative Promoter of the Human Neuronal Nitric Oxide Synthase Gene Is Expressed Specifically in Leydig Cells

“…19 A PacI linker was inserted at the 3Ј HindIII site of placC/␣ 1 G. A 6.5-kb KpnI-MunI genomic DNA fragment containing 81-bp 5Ј-UTR sequence and 6.5-kb 5Ј-flanking regions of the human TnNOS was obtained from a human genomic DNA subclone B42R/B-T1. 15 This 6.5-kb fragment containing the putative promoter for TnNOS was inserted into the KpnI-SalI site upstream of the LacZ open reading frame in placC/␣ 1 G to obtain plasmid Ϫ6500/ϩ81 pTn-NOS/lacC/␣ 1 G ( Figure 1A) placing the reporter gene under the transcriptional control of the human TnNOS 5Ј-flanking regions. The proximal portion (1.9 kb) of the 6.5-kb KpnI-MunI fragment has been previously reported (GenBank/EMBL Data Bank accession no.…”

“…Blots were incubated with anti-human nNOS monoclonal antibody directed against the COOH-terminus of the protein (1:500 dilution) (Transduction Laboratories) and subsequently with horseradish peroxidase-conjugated sheep anti-mouse secondary antibody (1:20,000 dilution) (Amersham, Arlington Heights, IL), as previously described. 15 Signal detection was facilitated with enhanced chemiluminescence (ECL, Amersham).…”

“…13 In a recent study we reported the cloning and characterization of a novel, testis-specific nNOS mRNA transcript (TnNOS) that accounted for approximately half of the total nNOS mRNA species expressed in the testis. 15 Transcription of TnNOS initiates from a novel noncoding downstream exon 1 (Tex 1) that is localized in intron 3 of the NOS1 gene. This exon is then spliced to another novel exon (Tex2) and then to exon 4 of the full-length nNOS.…”

“…Translation of the TnNOS variant transcripts produces an NH 2 -terminal truncated protein analogous to nNOS␥. 15,16 nNOS␥ represents a 125-kd protein expressed from exon 2-deleted full-length nNOS transcripts in human 15 and mouse. 16 When stably expressed in CHO-K1 cells, the 125-kd protein encoded by TnNOS possesses NOS enzymatic activity comparable to that of the full-length nNOS (160 kd), 15 although a comprehensive understanding of the biochemistry of this NOS variant is awaited.…”

“…15,16 nNOS␥ represents a 125-kd protein expressed from exon 2-deleted full-length nNOS transcripts in human 15 and mouse. 16 When stably expressed in CHO-K1 cells, the 125-kd protein encoded by TnNOS possesses NOS enzymatic activity comparable to that of the full-length nNOS (160 kd), 15 although a comprehensive understanding of the biochemistry of this NOS variant is awaited. TnNOS may have a unique biological role in the testis given that the protein domain implicated in functional interaction with the protein inhibitor of nNOS (PIN), which is highly expressed in this organ, 15,17 is removed in this NH 2 -deleted nNOS variant.…”