Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in S. cerevisiae, we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components. This revealed three control mechanisms for pA tail length. First, we found that the pA binding protein (PABP) Nab2p is the primary regulator of pA tail length. Second, when Nab2p is limiting, the nuclear pool of Pab1p, the second major PABP in yeast, controls the process. Third, when both PABPs are absent, the cleavage and polyadenylation factor (CPF) limits pA tail synthesis. Thus, Pab1p and CPF provide fail-safe mechanisms to a primary Nab2p-dependent pathway, thereby preventing uncontrolled polyadenylation and allowing mRNA export and translation.

中文翻译：

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酿酒酵母 mRNA poly(A) 尾合成的三层控制

大多数真核 mRNA 的生物发生涉及通过切割和多聚腺苷酸化机制添加未模板化的多聚腺苷 (pA) 尾。pA 尾及其确切长度会影响 mRNA 稳定性、核输出和翻译。定义如何在酿酒酵母中控制多腺苷酸化，我们使用了一种能够评估核 pA 尾合成的体内测定，通过直接 RNA 测序分析尾长分布，并用纯化的成分重组聚腺苷酸化反应。这揭示了 pA 尾长的三种控制机制。首先，我们发现 pA 结合蛋白 (PABP) Nab2p 是 pA 尾长的主要调节因子。其次，当 Nab2p 受到限制时，酵母中第二个主要 PABP 的核池 Pab1p 控制该过程。第三，当两个 PABP 都不存在时，切割和多聚腺苷酸化因子 (CPF) 限制了 pA 尾的合成。因此，Pab1p 和 CPF 为主要的 Nab2p 依赖性途径提供故障保护机制，从而防止不受控制的多腺苷酸化并允许 mRNA 输出和翻译。