Michel Y M, Poncet D, Piron M, Kean K M, Borman A M
Unité de Génétique Moléculaire des Virus Respiratoires, CNRS URA 1966, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France.
J Biol Chem. 2000 Oct 13;275(41):32268-76. doi: 10.1074/jbc.M004304200.
The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.
真核生物信使核糖核酸（mRNA）的5′端帽子结构和3′端聚腺苷酸（poly(A)）尾巴协同作用，在体内能协同刺激翻译起始，这一现象迄今为止仅在含有内源性竞争mRNA的体外翻译系统中观察到。在此，我们描述了经核酸酶处理的兔网织红细胞裂解物和人宫颈癌细胞（HeLa）细胞质提取物，它们在不存在此类竞争RNA的情况下能重现帽子结构-poly(A)协同效应。通过超速离心使提取物依赖于poly(A)，以部分去除核糖体和相关起始因子。在最佳条件下，网织红细胞裂解物中的协同效应值接近10倍。利用该系统，我们研究了poly(A)刺激翻译的分子机制。最大的帽子结构-poly(A)协同作用需要真核起始因子4G-聚腺苷酸结合蛋白（eIF4G-PABP）相互作用的完整性，这表明协同效应源于mRNA环化。此外，多聚腺苷酸化刺激了无帽子结构的细胞mRNA翻译以及由脑心肌炎病毒内部核糖体进入位点（IRES）驱动的翻译。poly(A)的这些效应也对eIF4G-PABP相互作用的破坏敏感，这表明5′-3′端的相互作用在经典mRNA和含IRES的mRNA之间在功能上是保守的。最后，我们证明一种从eIF4G上驱逐PABP的轮状病毒非结构蛋白能够引发轮状病毒感染期间所见的宿主细胞翻译关闭。
