Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0600, USA.
J Biol Chem. 2012 Oct 19;287(43):36370-83. doi: 10.1074/jbc.M112.373522. Epub 2012 Sep 6.
PUF proteins are a conserved family of eukaryotic RNA-binding proteins that regulate specific mRNAs: they control many processes including stem cell proliferation, fertility, and memory formation. PUFs repress protein expression from their target mRNAs but the mechanism by which they do so remains unclear, especially for humans. Humans possess two PUF proteins, PUM1 and PUM2, which exhibit similar RNA binding specificities. Here we report new insights into their regulatory activities and mechanisms of action. We developed functional assays to measure sequence-specific repression by PUM1 and PUM2. Both robustly inhibit translation and promote mRNA degradation. Purified PUM complexes were found to contain subunits of the CCR4-NOT (CNOT) complex, which contains multiple enzymes that catalyze mRNA deadenylation. PUMs interact with the CNOT deadenylase subunits in vitro. We used three approaches to determine the importance of deadenylases for PUM repression. First, dominant-negative mutants of CNOT7 and CNOT8 reduced PUM repression. Second, RNA interference depletion of the deadenylases alleviated PUM repression. Third, the poly(A) tail was necessary for maximal PUM repression. These findings demonstrate a conserved mechanism of PUF-mediated repression via direct recruitment of the CCR4-POP2-NOT deadenylase leading to translational inhibition and mRNA degradation. A second, deadenylation independent mechanism was revealed by the finding that PUMs repress an mRNA that lacks a poly(A) tail. Thus, human PUMs are repressors capable of deadenylation-dependent and -independent modes of repression.
PUF 蛋白是真核生物 RNA 结合蛋白的一个保守家族，可调节特定的 mRNA：它们控制着许多过程，包括干细胞增殖、生育能力和记忆形成。PUF 从其靶 mRNA 抑制蛋白质表达，但它们的作用机制尚不清楚，尤其是对人类而言。人类拥有两种 PUF 蛋白，PUM1 和 PUM2，它们表现出相似的 RNA 结合特异性。在这里，我们报告了对其调节活性和作用机制的新见解。我们开发了功能测定法来测量 PUM1 和 PUM2 的序列特异性抑制作用。两者都能强烈抑制翻译并促进 mRNA 降解。纯化的 PUM 复合物被发现含有 CCR4-NOT（CNOT）复合物的亚基，该复合物包含多个催化 mRNA 去腺苷酸化的酶。PUM 与体外的 CNOT 脱腺苷酸酶亚基相互作用。我们使用三种方法来确定脱腺苷酸酶对 PUM 抑制的重要性。首先，CNOT7 和 CNOT8 的显性负突变减少了 PUM 的抑制作用。其次，脱腺苷酸酶的 RNA 干扰耗竭减轻了 PUM 的抑制作用。第三，多聚（A）尾对于最大 PUM 抑制是必需的。这些发现证明了通过直接募集 CCR4-POP2-NOT 脱腺苷酸酶来抑制 PUF 介导的抑制的保守机制，导致翻译抑制和 mRNA 降解。第二个脱腺苷酸化独立的机制是通过发现 PUM 抑制缺乏多聚（A）尾的 mRNA 而揭示的。因此，人类 PUM 是能够进行脱腺苷酸化依赖和非依赖模式抑制的抑制剂。
