Post-transcriptional factors importantly contribute to the rapid and coordinated expression of the multiple genes required for the adaptation of living organisms to environmental stresses. In the model eukaryote Saccharomyces cerevisiae, a conserved mRNA-binding protein, known as Cth2, modulates the metabolic response to iron deficiency. Cth2 is a tandem zinc-finger (TZF)-containing protein that co-transcriptionally binds to adenine/uracil-rich elements (ARE) present in the 3′-untranslated region of iron-related mRNAs to promote their turnover. The nuclear binding of Cth2 to mRNAs via its TZFs is indispensable for its export to the cytoplasm. Although Cth2 nucleocytoplasmic transport is essential for its regulatory function, little is known about the recruitment of the mRNA degradation machinery. Here, we investigate the sequential assembly of mRNA decay factors during Cth2 shuttling. By using an enzymatic in vivo proximity assay called M-track, we show that Cth2 associates to the RNA helicase Dhh1 and the deadenylase Pop2/Caf1 before binding to its target mRNAs. The recruitment of Dhh1 to Cth2 requires the integrity of the Ccr4-Pop2 deadenylase complex, whereas the interaction between Cth2 and Pop2 needs Ccr4 but not Dhh1. M-track assays also show that Cth2-binding to ARE-containing mRNAs is necessary for the interaction between Cth2 and the exonuclease Xrn1. The importance of these interactions is highlighted by the specific growth defect in iron-deficient conditions displayed by cells lacking Dhh1, Pop2, Ccr4 or Xrn1. These results exemplify the stepwise process of assembly of different mRNA decay factors onto an mRNA-binding protein during the mechanism of post-transcriptional regulation.

转录后因子重要地促进了使活生物体适应环境压力所需的多个基因的快速协调表达。在真核生物酿酒酵母模型中，一种保守的mRNA结合蛋白，称为Cth2，可调节对铁缺乏的代谢反应。Cth2是含串联锌指（TZF）的蛋白质，可与铁相关mRNA 3'非翻译区中存在的腺嘌呤/富尿嘧啶的元素（ARE）共转录结合，从而促进其更新。Cth2通过以下方式与mRNA核结合它的TZF对于其向细胞质的出口是必不可少的。尽管Cth2核质转运对其调节功能至关重要，但对募集mRNA降解机制的了解很少。在这里，我们研究了Cth2穿梭过程中mRNA衰减因子的顺序组装。通过在体内使用酶称为M-track的邻近测定法，我们显示Cth2在结合至其目标mRNA之前先与RNA解旋酶Dhh1和去烯基酶Pop2 / Caf1相关。将Dhh1募集到Cth2需要Ccr4-Pop2腺苷酸酶复合物的完整性，而Cth2和Pop2之间的相互作用需要Ccr4，但不需要Dhh1。M-track检测还表明，Cth2与含ARE的mRNA结合对于Cth2和核酸外切酶Xrn1之间的相互作用是必需的。这些相互作用的重要性通过缺乏Dhh1，Pop2，Ccr4或Xrn1的细胞在缺铁条件下出现的特定生长缺陷而凸显出来。这些结果说明了转录后调控机制中，将不同的mRNA衰减因子组装到mRNA结合蛋白上的逐步过程。