Mazumder B, Seshadri V, Imataka H, Sonenberg N, Fox P L
Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Mol Cell Biol. 2001 Oct;21(19):6440-9. doi: 10.1128/MCB.21.19.6440-6449.2001.
Ceruloplasmin (Cp) 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 Cp 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 Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 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 poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp 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 luciferase reporter upstream of the Cp 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 Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor 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.
铜蓝蛋白（Cp）是一种由肝脏和单核细胞分泌的糖蛋白，可能在炎症和铁代谢中发挥作用。我们之前表明，γ干扰素（IFN-γ）可诱导人U937单核细胞合成Cp，但随后该合成通过一种转录本特异性的翻译沉默机制而停止，该机制涉及一种胞质因子与Cp mRNA 3'非翻译区（UTR）的结合。为了研究Cp 3'-UTR处的蛋白质相互作用如何抑制远处5'端的翻译起始，我们考虑了mRNA翻译的“闭环”模型。在该模型中，转录本末端通过多聚腺苷酸结合蛋白（PABP）与多聚腺苷酸尾和起始因子eIF4G的相互作用而聚集在一起。使用兔网织红细胞裂解物中的嵌合报告转录本测试了这些元件对Cp翻译控制的影响。由于来自IFN-γ处理细胞的胞质抑制剂对Cp 3'-UTR上游的荧光素酶报告基因的翻译抑制作用最小，但几乎完全阻断了含有多聚腺苷酸尾的转录本的翻译，因此显示了对多聚腺苷酸的需求。同样，对内源性Cp mRNA的沉默也显示了对多聚腺苷酸的需求。我们考虑了胞质抑制剂阻断PABP与多聚腺苷酸尾或与eIF4G相互作用的可能性。我们发现这些相互作用均未受到抑制，通过对PABP进行免疫沉淀，随后通过逆转录-PCR对多聚腺苷酸尾进行定量以及通过免疫印迹分析对eIF4G进行定量来证明。我们考虑了另一种可能性，即这些相互作用是翻译沉默所必需的。当用抗人PABP抗体从网织红细胞裂解物中耗尽PABP时，胞质因子不会抑制嵌合报告基因的翻译，从而表明对PABP的需求。同样，在用抗人eIF4G抗体处理的裂解物中，胞质提取物不会抑制嵌合报告基因的翻译，从而表明对eIF4G的需求。这些数据表明，Cp的翻译沉默需要mRNA环化的三个基本元件多聚腺苷酸、PABP和eIF4G的相互作用。我们认为，Cp mRNA环化使胞质Cp 3'-UTR结合因子靠近翻译起始位点，在那里它通过一种未确定的机制使翻译沉默。这些结果表明，转录本环化除了在提高翻译效率方面具有重要功能外，还可能作为转录本特异性翻译控制的一个基本结构决定因素。
