Translational Regulation of Human Neuronal Nitric-oxide Synthase by an Alternatively Spliced 5′-Untranslated Region Leader Exon

Abstract

Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

Single-nucleotide promoter polymorphism alters transcription of neuronal nitric oxide synthase exon 1c in infantile hypertrophic pyloric stenosis

“…No novel 5Ј-mRNA forms were found. In addition, we could not detect the alternatively spliced untranslated leader exon AS in infantile pyloric specimens, which has been shown to alter translation of human nNOS (26).…”

“…The nNOS gene is one of the most complex genes known in terms of first-exon usage and alternative splicing (24). Nine distinct first-exon transcripts (exon 1a-1i), generated by alternative promoter usage, are present in the human gut, leading to nNOS mRNA variants with different 5Ј-untranslated regions and translational efficiencies (23)(24)(25)(26)(27). Cell-and sitespecific expression patterns (23,25) indicate a spatial and temporal regulation of nNOS expression under different physiological or pathological conditions.…”

Comparative analysis of human NK cell activation induced by NKG2D and natural cytotoxicity receptors

“…Yet, alternative splicing excluding the 5' noncoding exon 2 and 3 of hNKG2D were reproducibly detected, and we cannot exclude that these alternative splicing events provide a peculiar RNA structural context that favors the initiation of translation from hNKG2D ATG2. An impact of alternative splicing of the 5' non coding region in the control of the alternative usage of ATG translation initiation codons has been demonstrated for other genes as a tuning mechanism for protein isoform production [26]. Alternatively, since the kinetics of induction of NKG2D-S transcripts in mice are restrained to a narrow time-window during the course of NK cell activation [12], we cannot exclude that our search for transcripts in activated human NK cells and +ˇT cells missed the time-points at which a putative, short human NKG2D transcript could be detected.…”

An alternatively‐spliced exon in the 5′‐UTR of human ALAS1 mRNA inhibits translation and renders it resistant to haem‐mediated decay

“…The process of mRNA turnover is intimately linked to translation and numerous 5 0 -UTRs have been shown to regulate translation through mRNA-specific characteristics such as size, GC content, secondary and tertiary structure, and the location of potentially active upstream open reading frames (uORFs) [17], e.g., translation of nitric oxide synthase has been shown to be regulated by an alternatively spliced 5 0 -UTR [24]. The ALAS1 minor 5 0 -UTR sequence exhibits a number of features that might be responsible for its reduced translation.…”