The Nitric Oxide Cycle: Translational Regulation of Argininosuccinate Synthase by an Upstream Open Reading Frame

Abstract

Argininosuccinate synthase (AS) catalyzes the rate-limiting step in the recycling of citrulline to arginine, which in endothelial cells, is tightly coupled to the production of nitric oxide. Previous work has established that endothelial AS mRNA can be initiated from multiple start sites, generating mRNAs with different length, overlapping 5′-UTRs. Nearly 10% of AS transcripts are longer variants that contain an out-of-frame, upstream open reading frame (uORF). Overexpression of this uORF suppresses AS expression while selective knockdown of the longer transcripts results in increased AS protein expression. To investigate the role of the AS uORF in the regulation of AS translation polysome profile analyses were performed. Real time RT-PCR quantitation of RNA isolated from sucrose gradient fractions showed a substantial reduction of AS mRNA associated with ribosomes across the gradient when the AS uORF was overexpressed in endothelial cells as compared to the overexpression of a frame shifted mutant protein identical in size. The largest decrease in AS mRNA was observed in the polysome region. GAPDH mRNA distribution and levels were very similar under both conditions. To further support the role of the AS uORF in the regulation of AS translation, in vitro translation of AS mRNA in the presence of AS uORF protein showed a marked decrease in the translation of AS. Taken together, these results indicate that the AS uORF plays a significant role in the translational attenuation of AS expression in endothelial cells. (Supported by the AHA Grant 0455228B)