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

Abstract

Argininosuccinate synthase (AS) catalyzes the ratelimiting 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. Nitric oxide (NO)1 synthesized from arginine by endothelial nitric-oxide synthase is a potent vasodilator and a critical modulator of blood flow and blood pressure. In addition, it mediates vasoprotective actions through inhibiting smooth muscle proliferation, platelet aggregation, and leukocyte adhesion (1-3). Under pathophysiological conditions associated with endothelial dysfunction, such as heart failure (4), hypertension, hypercholesterolemia, atherosclerosis (5), and diabetes (6), the ability to produce NO seems to be impaired. Paradoxically, NO production can be impaired by limited availability of the substrate arginine, despite apparently saturating levels of intracellular and extracellular arginine (7-10). We have previously shown that under normal conditions, the essential arginine available for NO production is derived from the recycling of citrulline to arginine, catalyzed by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase (AL) (11,12). Although these two enzymes have been studied extensively in liver, where they participate in the urea cycle (13), it was not until the discovery of NO that their function in non-hepatic tissues was clarified. In endothelial cells, AS and AL play a critical role in the operation of a citrulline-NO cycle, which supports endothelial NO production (14 -17).Because AS catalyzes the rate-limiting step in the citrulline-NO cycle (15), our initial studies have focused on the molecular basis for the functional role of endothelial AS. Endothelial and hepatic AS appear to have the same ...

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

“…The polycistronic nature of AltORF encoding mRNAs can potentially lead to intriguing functional interplays between reference and alternative proteins, such as direct interaction between the reference and alternative proteins [11], [49]. There is also evidence that an upstream ORF not only regulates the expression of a downstream RefORF by interfering in cis with canonical AUG recognition by scanning ribosomes, but also reduces the translational efficiency of the RefORF in trans [45], [50]. AltORFs translation products could also be of particular importance during the thymal selection of T lymphocytes, serving as “cryptic T-cell epitopes”.…”

Tumor necrosis factor-α reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

“…This strategy takes advantage of a NcoI site within the luciferase gene, close to the start codon, to allow for the AS 5Ј-UTR to be cloned adjacent to the start codon. For mutant constructs, mutations were made in the three Sp1 sites in the p3ASP189 construct using a three-way PCR method (34). Primers AS189mut1 (5Ј-CTCCAGGCGGGTTCCGGGCCCGGG-3Ј), AS189mut2 (5Ј-CCGGGTTCGGGGTCTGTGGC-3Ј), and AS189mut3 (5Ј-AC-CGGACCTGACCCCGGG-3Ј) were combined with ASRluc to amplify fragments that contained the mutations.…”

A perspective on mammalian upstream open reading frame function

“…The major transcripts (present at >90% of all AS transcripts within a cell) contain a short UTR with no uAUG, whereas the longer UTRs of the minor products contain an inhibitory uORF (Pendleton et al, 2002). Transfection of the AS uORF construct into bovine aortic endothelial cells repressed endogenous AS protein expression, with mutational analysis demonstrating that both the uORF peptide sequence and length being essential determinants of repression (Pendleton et al, 2005). Other examples of trans-repression have thus far been demonstrated by the addition of high concentrations of uORF peptides within cell-free systems.…”