
<ArticleTitle>Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, <prot>poly(A)-binding protein</prot>, and <prot>eukaryotic translation initiation factor 4G</prot>.</ArticleTitle>
<AbstractText><prot>Ceruloplasmin</prot> (<prot>Cp</prot>) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced <prot>Cp</prot> synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the <prot>Cp</prot> mRNA 3' untranslated region (UTR). To investigate how protein interactions at the <prot>Cp</prot> 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of <prot>poly(A)-binding protein</prot> (<prot>PABP</prot>) with both the poly(A) tail and initiation factor <prot>eIF4G</prot>. The effect of these elements on <prot>Cp</prot> translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a <prot>luciferase</prot> reporter upstream of the <prot>Cp</prot> 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous <prot>Cp</prot> mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of <prot>PABP</prot> with the poly(A) tail or with <prot>eIF4G</prot>. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of <prot>PABP</prot> followed by quantitation of the poly(A) tail by reverse transcription-PCR and of <prot>eIF4G</prot> by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When <prot>PABP</prot> was depleted from the reticulocyte lysate with anti-human <prot>PABP</prot> antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for <prot>PABP</prot>. Similarly, in lysates treated with anti-human <prot>eIF4G</prot> antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for <prot>eIF4G</prot>. These data show that translational silencing of <prot>Cp</prot> requires interactions of three essential elements of mRNA circularization, poly(A), <prot>PABP</prot>, and <prot>eIF4G</prot>. We suggest that <prot>Cp</prot> mRNA circularization brings <prot>the cytosolic <prot>Cp</prot> 3'-UTR-binding factor</prot> into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.</AbstractText>
