Lanterna Rally

Author: Derek Colin Newton

Publisher: National Library of Canada = Bibliothèque nationale du Canada

Published: 2004

Total Pages: 644

ISBN-13: 9780612917705

Protein synthesis is the ultimate goal of gene expression and a key control point for regulation. In particular, it enables the cell to rapidly manipulate protein production without new mRNA synthesis, processing or export. The human neuronal nitric oxide synthase (nNOS) gene is an excellent model to study the regulated translation of a mRNA to protein. Numerous and distinct nNOS mRNAs are generated from a single gene locus as a result of alternate promoter usage and alternate splicing patterns. A comprehensive analysis of the structure of neuronal NOS (nNOS) mRNA species revealed NOS1 to be one of the most structurally diverse human genes described to date in terms of promoter usage. Unique exon 1 variants are utilized for transcript initiation in diverse tissues and this dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. These highly structured nNOS mRNA 5'-untranslated regions (5'-UTRs) were demonstrated to have profound effects on translation in both in vitro assays and cells and to contain cis-RNA elements that modulate translational efficiency in response to changes in cellular phenotype. In addition to promoter diversity, the human nNOS gene contains an alternately spliced (AS) exon present within the 5' -UTR. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore we assessed its functional relevance. Transient and stable transfection methods revealed that this alternatively spliced exon acts as a potent translational repressor. Hence a unique splicing event within a 5'-UTR is demonstrated to introduce a translational control element. Importantly, this represented a newer model for the translational control in mammalian cells. Mutational analysis identified a putative stem-loop structure within this exon that acts as the functional cis-element. Subsequent RNA-EMSA analyses revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. An affinity purification scheme was developed and the cytoskeletal proteins actin and alpha-actinin were identified as components of the ribonucleoprotein complex associated with this exon. This intriguing finding indicates that this exon acts as a subcellular localization signal for select nNOS mRNAs.