Oct 31, 2002

Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon

The Journal of Biological Chemistry
Derek C NewtonP A Marsden

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 s...Continue Reading

Mentioned in this Paper

Nested Transcripts
Transcriptional Regulation
RNA Conformation
Transfection
Untranslated Regions
Transcription Repressor/Corepressor
Exons
NOS1 protein, human
Cytoplasmic RNA
Nos1 protein, rat

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