The 3' to 5' exonuclease Pop2p (Caf1p) is part of the CCR4-NOT deadenylation complex that removes poly(A) tails from mRNAs in cells. Pop2p is structurally conserved in eukaryotes, but S. cerevisiae Pop2p harbors non-canonical amino acids in its catalytic center. The enzymatic properties of S. cerevisiae Pop2p are not well defined. Here we characterize the RNA exonuclease activity of recombinant S. cerevisiae Pop2p. We find that S. cerevisiae Pop2p degrades RNAs via two alternative reactions pathways, one generating nucleotides with 5'- phosphates and RNA intermediates with 3'-hydroxyls, and the other generating nucleotides with 3'-phosphates and RNA intermediates with 3'-phosphates. The enzyme is not able to initiate the reaction on RNAs with a 3'-phosphate, which leads to accumulation of RNAs with 3'-phosphates that can exceed 10 nt and are resistant to further degradation by S. cerevisiae Pop2p. We further demonstrate that S. cerevisiae Pop2p degrades RNAs in three reaction phases; an initial distributive phase, a second processive phase and a third phase during which processivity gradually declines. We also show that mutations of subsets of amino acids in the catalytic center, including those previously thought to inactivate the enzyme, moderately reduce, but not eliminate activity. Only mutation of all five amino acids in the catalytic center diminishes activity of Pop2p to background levels. Collectively, our results reveal robust exonuclease activity of S. cerevisiae Pop2p with unusual enzymatic properties, characterized by alternative degradation pathways, multiple reaction phases and functional redundancy of amino acids in the catalytic core.

中文翻译：

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来自酿酒酵母的外切核酸酶 Pop2p 的替代 RNA 降解途径

3' 到 5' 外切核酸酶 Pop2p (Caf1p) 是 CCR4-NOT 去腺苷酸复合物的一部分，可从细胞中的 mRNA 中去除 poly(A) 尾。Pop2p 在真核生物中结构保守，但酿酒酵母 Pop2p 在其催化中心含有非规范氨基酸。S. cerevisiae Pop2p 的酶学特性没有明确定义。在这里，我们描述了重组酿酒酵母 Pop2p 的 RNA 核酸外切酶活性。我们发现 S. cerevisiae Pop2p 通过两种替代反应途径降解 RNA，一种产生具有 5'-磷酸的核苷酸和具有 3'-羟基的 RNA 中间体，另一种产生具有 3'-磷酸的核苷酸和具有 3'-磷酸的 RNA 中间体. 该酶不能启动带有 3'-磷酸的 RNA 的反应，这会导致带有 3' 的 RNA 积累 -磷酸盐，可超过 10 nt，并能抵抗 S. cerevisiae Pop2p 的进一步降解。我们进一步证明 S. cerevisiae Pop2p 在三个反应阶段降解 RNA；初始分配阶段，第二个过程阶段和第三个阶段，在此期间过程性逐渐下降。我们还表明，催化中心氨基酸亚群的突变，包括以前认为使酶失活的那些突变，会适度降低，但不会消除活性。只有催化中心所有五个氨基酸的突变才会将 Pop2p 的活性降低到背景水平。总的来说，我们的结果揭示了 S. cerevisiae Pop2p 具有不寻常的酶特性的强大的外切核酸酶活性，其特征在于替代降解途径，