Transcript-selective translational silencing by gamma interferon is directed by a novel structural element in the ceruloplasmin mRNA 3' untranslated region.

Ceruloplasmin (Cp) is a multifunctional, copper-containing glycoprotein produced by the liver and secreted into the plasma. As an acute-phase protein, its plasma concentration increases up to twofold during multiple inflammatory conditions. Plasma Cp has been reported to be an independent risk factor for cardiovascular disease, including atherosclerosis, carotid restenosis after endarterectomy, and myocardial infarction (24, 36). Several laboratories have shown that Cp copper can cause oxidative modification of low density lipoprotein, and this activity may contribute to a direct role of Cp in the pathogenesis of atherosclerosis (7, 26, 39). Cp also has a ferroxidase activity thought to be necessary for optimal loading of iron into transferrin (27). The important physiological role for Cp in iron homeostasis has been convincingly demonstrated by finding iron overload in humans with hereditary Cp deficiency (40) and in mice with targeted Cp gene disruption (9). In addition to its synthesis by hepatic cells, Cp is synthesized and secreted by activated monocyte/macrophages. Treatment of human monocytic U937 cells or peripheral blood monocytes with gamma interferon (IFN-γ) induces the expression of both Cp mRNA and protein (19). Our laboratory has shown that induced expression of Cp by IFN-γ is subject to a unique transcript-selective translational silencing mechanism in which synthesis is terminated after about 16 h, even in the presence of abundant Cp mRNA (18). Our results suggest the presence of a cytosolic inhibitor, since lysates from U937 cells treated with IFN-γ for 24 h (but not for 8 h) inhibited in vitro translation of Cp in a reticulocyte lysate. The inhibition was accompanied by the binding of a trans-acting factor to the 247-nucleotide (nt), Cp 3′ untranslated region (3′-UTR), as shown by an RNA electrophoretic mobility shift assay (EMSA) using radiolabeled Cp 3′-UTR as probe and by an in vitro translation assay in which unlabeled Cp 3′-UTR, added in excess as a decoy, overcame the inhibition by cytosolic extracts (18). There are multiple examples of translational regulation directed by proteins interacting with the 3′-UTR (see references 32 and 34 for review). The mechanism by which 3′-UTR-binding proteins inhibit translation-initiation at the distant 5′-UTR is incompletely understood, but studies showing that mRNA may form a closed loop by 5′-to-3′ interactions may provide an important clue (10). This “circular” model has been helpful in understanding 3′-UTR-mediated translational silencing of Cp by IFN-γ (21). The mechanism of translational silencing was investigated by in vitro translation of a heterologous reporter transcript consisting of the luciferase (Luc) open reading frame (ORF) ahead of the full-length Cp 3′-UTR and a 30-nt poly(A) tail. Removal or inactivation of any of the components of transcript circularization, i.e., the poly(A) tail, eukaryotic initiation factor 4G, or poly(A)-binding protein (PABP), prevented the translational silencing activity (21). These results have led us to propose a mechanism of translational control in which interactions of the termini carry a 3′-interacting inhibitor protein (or complex) into the vicinity of the translation-initiation site where it can silence translation, possibly by binding to or interfering with an initiation factor or ribosomal protein (20, 21). Transcript-selective translational control generally is directed by a specific cis element characterized by a requirement for specific structural features as well as for invariant sequences. In our previous study, a limited deletion analysis of the Cp 3′-UTR showed that the binding site of the translational inhibitor was present in overlapping UTR fragments 51-247 and 1-150 [where position 1 is the first nucleotide after the stop codon and 247 is the last nucleotide before the poly(A) tail in the short form of the Cp 3′-UTR] (18). This result suggested that the required element was contained in the UTR region 51-150 common to both fragments. Computational folding of the full-length Cp 3′-UTR indicates that the element is located within an elongated structure comprising multiple stems and loops (see Fig. ​Fig.1A).1A). We here describe a detailed analysis of the Cp 3′-UTR and provide evidence for a novel structural element that mediates the inhibition of Cp translation in IFN-γ-activated monocytic cells. FIG. 1. Location of the translational silencing element in the human Cp 3′-UTR. (A) Folding structure of the full-length Cp 3′-UTR as determined by the mfold algorithm. The terminal positions of key constructs are indicated by boxes. (B) A summary ...