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J. Biol. Chem., Vol. 280, Issue 25, 24252-24260, June 24, 2005

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Regulation of Endothelial Argininosuccinate Synthase Expression and NO Production by an Upstream Open Reading Frame^*

Laura C. Pendleton, Bonnie L. Goodwin, Larry P. Solomonson, and Duane C. Eichler

From the Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa, Florida 33612

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 (NO). In previous work, we established that endothelial AS mRNA can be initiated from multiple start sites, generating co-expressed mRNA variants with different 5'-untranslated regions (5'-UTRs). One of the 5'-UTRs, the shortest form, represents greater than 90% of the total AS mRNA. Two other extended 5'-UTR forms of AS mRNA, resulting from upstream initiations, contain an out-of-frame, upstream open reading frame (uORF). In this study, the function of the extended 5'-UTRs of AS mRNA was investigated. Single base insertions to place the uORF in-frame, and mutations to extend the uORF, demonstrated functionality, both in vitro with AS constructs and in vivo with luciferase constructs. Overexpression of the uORF suppressed endothelial AS protein expression, whereas specific silencing of the uORF AS mRNAs resulted in the coordinate up-regulation of AS protein and NO production. Expression of the full-length of the uORF was necessary to mediate a trans-suppressive effect on endothelial AS expression, demonstrating that the translation product itself affects regulation. In conclusion, the uORF found in the extended, overlapping 5'-UTR AS mRNA species suppresses endothelial AS expression, providing a novel mechanism for regulating endothelial NO production by limiting the availability of arginine.

Received for publication, January 4, 2005 , and in revised form, April 19, 2005.

^* This work was supported by the USF Research Foundation Mary and Walter Traskiewicz Memorial Fund and the American Heart Association, Florida Affiliate Grant 0455228B. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of South Florida, 12901 Bruce B. Downs Blvd., MDC 7, Tampa, FL 33612. Tel.: 813-974-9716; Fax: 813-974-9350; E-mail: deichler{at}hsc.usf.edu.

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