Goldstrohm Aaron C, Seay Daniel J, Hook Brad A, Wickens Marvin
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
J Biol Chem. 2007 Jan 5;282(1):109-14. doi: 10.1074/jbc.M609413200. Epub 2006 Nov 7.
PUF proteins control gene expression by binding to the 3'-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.
PUF蛋白通过与特定mRNA的3'非翻译区结合并触发mRNA降解或翻译抑制来控制基因表达。在此，我们聚焦于PUF介导的调控机制。酵母PUF蛋白Mpt5p可调控HO mRNA并促进其poly(A)尾的去除（即去腺苷酸化）。体内Mpt5p的抑制作用依赖于POP2，它是细胞质中使mRNA去腺苷酸化的Ccr4p-Pop2p-Not复合物的一个组分。在本研究中，我们阐明了Ccr4p和Pop2p去腺苷酸化酶在Mpt5p调控的去腺苷酸化过程中的各自作用。在体内和体外，Pop2p和Ccr4p蛋白都是Mpt5p调控的HO去腺苷酸化所必需的。然而，这两种蛋白的需求差异极大：Ccr4p的酶活性至关重要，而Pop2p的酶活性则并非必需。我们得出结论，Pop2p是一个桥梁，PUF蛋白通过它将Ccr4p酶招募至靶mRNA，从而促进去腺苷酸化。我们的数据表明，PUF蛋白可能通过依赖去腺苷酸化和不依赖去腺苷酸化的机制来增强mRNA降解并抑制表达，利用同一个Pop2p桥梁将多功能Pop2p复合物招募至mRNA。
