Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.
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.
Newton
Derek C
DC
Renal Division and the Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Bevan
Sian C
SC
Choi
Stephen
S
Robb
G Brett
GB
Millar
Adam
A
Wang
Yang
Y
Marsden
Philip A
PA
D020121:5' Untranslated Regions / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005091:Exons / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012315:RNA Caps; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014018:Tissue Distribution
D020121:5' Untranslated Regions; D012315:RNA Caps; D012333:RNA, Messenger; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; C496322:Nos1 protein, rat