Department of Molecular Biology and Genetics, Centre for mRNP Biogenesis and Metabolism, Aarhus University, Gustav Wieds Vej 10c and C. F. Møllers Allé 130, DK-8000 Aarhus C, Denmark.
Nucleic Acids Res. 2012 Jan;40(2):837-46. doi: 10.1093/nar/gkr782. Epub 2011 Sep 29.
Deadenylation is the first and rate-limiting step during turnover of mRNAs in eukaryotes. In the yeast, Saccharomyces cerevisiae, two distinct 3'-5' exonucleases, Pop2p and Ccr4p, have been identified within the Ccr4-NOT deadenylase complex, belonging to the DEDD and Exonuclease-Endonuclease-Phosphatase (EEP) families, respectively. Ngl3p has been identified as a new member of the EEP family of exonucleases based on sequence homology, but its activity and biological roles are presently unknown. Here, we show using in vitro deadenylation assays on defined RNA species mimicking poly-A containing mRNAs that yeast Ngl3p is a functional 3'-5' exonuclease most active at slightly acidic conditions. We further show that the enzyme depends on divalent metal ions for activity and possesses specificity towards poly-A RNA similar to what has been observed for cellular deadenylases. The results suggest that Ngl3p is naturally involved in processing of poly-adenylated RNA and provide insights into the mechanistic variations observed among the redundant set of EEP enzymes found in yeast and higher eukaryotes.
去腺苷酸化是真核生物 mRNA 周转过程中的第一步和限速步骤。在酵母 Saccharomyces cerevisiae 中，两个不同的 3'-5' 外切核酸酶 Pop2p 和 Ccr4p 已在 Ccr4-NOT 脱腺苷酸酶复合物中被鉴定出来，分别属于 DEDD 和 Exonuclease-Endonuclease-Phosphatase (EEP) 家族。Ngl3p 基于序列同源性被鉴定为 EEP 家族的新成员，但它的活性和生物学作用目前尚不清楚。在这里，我们使用体外去腺苷酸化测定法在模拟含有 poly-A 的 mRNA 的定义 RNA 物种上进行实验，结果表明酵母 Ngl3p 是一种具有功能的 3'-5' 外切核酸酶，在略酸性条件下最活跃。我们进一步表明，该酶的活性依赖于二价金属离子，并且对 poly-A RNA 具有特异性，类似于已观察到的细胞脱腺苷酸酶。这些结果表明 Ngl3p 天然参与 poly-adenylated RNA 的加工，并深入了解在酵母和高等真核生物中发现的冗余 EEP 酶组中观察到的机制变化。
