Wwp2-mediated ubiquitination of the RNA polymerase II large subunit in mouse embryonic pluripotent stem cells
Wwp2-mediated ubiquitination of the RNA polymerase II large subunit in mouse embryonic pluripotent stem cells
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DOI：
10.1128/mcb.01667-06
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发表时间：
2007-08-01
影响因子：
5.3
通讯作者：
Jin, Ying
中科院分区：
文献类型：
--
作者：
Li, Hui;Zhang, Zhihong;Jin, Ying
泛素化和RNA聚合酶H大亚基Rpb1的降解不仅与DNA损伤诱导的停滞有关，还与其他转录障碍事件有关。然而，在哺乳动物细胞中，负责DNA损伤独立过程的泛素连接酶仍有待鉴定。在这里，我们鉴定了WWP2，一个小鼠Hect结构域泛素E3连接酶，是Rpb1的一个新的泛素连接酶。我们发现WWP2与小鼠Rpb1特异地相互作用，并在体外和体内针对它的泛素化。有趣的是，与Rpb1的相互作用和泛素化既不依赖于它的磷酸化状态，也不依赖于DNA损伤。然而，WWP2的酶活性是其对Rpb1进行泛素修饰所必需的。此外，我们的研究表明，WWP2和Rpb1之间的相互作用分别通过WWP2的WW结构域和Rpb1的C末端结构域来调节。值得注意的是，WWP2表达的下调影响了Rpb1的泛素化，并显著提高了其细胞内稳态蛋白水平。重要的是，我们鉴定了Rpb1 C-末端结构域的6个赖氨酸残基是由WWP2介导的泛素受体位点。这些结果表明，WWP2在正常生理条件下对Rpb1的表达起着重要的调节作用。
Ubiquitination and the degradation of the large subunit of RNA polymerase H, Rpb1, is not only involved in DNA damage-induced arrest but also in other transcription-obstructing events. However, the ubiquitin ligases responsible for DNA damage-independent processes in mammalian cells remain to be identified. Here, we identified Wwp2, a mouse HECT domain ubiquitin E3 ligase, as a novel ubiquitin ligase of Rpb1. We found that Wwp2 specifically interacted with mouse Rpb1 and targeted it for ubiquitination both in vitro and in vivo. Interestingly, the interaction with and ubiquitination of Rpb1 was dependent neither on its phosphorylation state nor on DNA damage. However, the enzymatic activity of Wwp2 was absolutely required for its ubiquitin modification of Rpb1. Furthermore, our study indicates that the interaction between Wwp2 and Rpb1 was mediated through WW domain of Wwp2 and C-terminal domain of Rpb1, respectively. Strikingly, downregulation of Wwp2 expression compromised Rpb1 ubiquitination and elevated its intracellular steady-state protein level significantly. Importantly, we identified six lysine residues in the C-terminal domain of Rpb1 as ubiquitin acceptor sites mediated by Wwp2. These results indicate that Wwp2 plays an important role in regulating expression of Rpb1 in normal physiological conditions.