生物谷报道：RNA聚合酶II大亚基（Rpb1）是决定真核生物信使RNA（mRNA）转录起始和延伸的最主要的功能亚基，存在多种翻译后修饰。其中，它的泛素化修饰和降解，不仅发生在DNA损伤引起的转录停滞过程中，而且涉及到其他的转录障碍事件。然而，在哺乳动物细胞中，在非DNA损伤依赖过程中发挥作用的Rpb1的泛素连接酶还没有被发现。健康科学研究所金颖研究员领导的分子发育生物学实验室的最新研究表明Wwp2，小鼠的HECT家族的泛素E3连接酶，是Rpb1新的泛素连结酶。研究结果显示在体内和体外，小鼠的Wwp2均能特异性结合Rpb1并催化它的泛素化修饰。有趣的是，Wwp2 对Rpb1的结合和泛素化修饰既不依赖于Rpb1的磷酸化状态也不依赖于DNA损伤。然而Wwp2的酶活性对于Rpb1的泛素化修饰是必需的。进一步的研究还显示，Wwp2和Rpb1的相互作用是通过Wwp2 的WW 结构域和Rpb1的C端结构域CTD介导的。当下调Wwp2的表达水平时，Rpb1的泛素化水平也随之降低，而蛋白水平则明显升高。通过质谱分析，Rpb1 CTD结构域中的6个赖氨酸残基被确认为Wwp2介导的泛素化位点。这些结果显示Wwp2在Rpb1正常生理状态下的表达调节中发挥重要功能。
该研究成果发表在07年8月的《分子细胞生物学》杂志上。（援引健康科学研究所）
原始出处:
Molecular and Cellular Biology, August 2007, p. 5296-5305, Vol. 27, No. 15
0270-7306/07/$08.00+0 doi:10.1128/MCB.01667-06
Wwp2-Mediated Ubiquitination of the RNA Polymerase II Large Subunit in Mouse Embryonic Pluripotent Stem Cells
Hui Li,1,2,5 Zhihong Zhang,1 Beibei Wang,1 Junmei Zhang,3 Yingming Zhao,3 and Ying Jin1,2,4*
Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, 225 South Chongqing Road, Shanghai 200025, China,1 Laboratory of Molecular Developmental Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, China,2 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390,3 Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China,4 Graduate School of Chinese Academy of Sciences, Beijing, China5
Received 6 September 2006/ Returned for modification 15 November 2006/ Accepted 13 May 2007
Ubiquitination and the degradation of the large subunit of RNA polymerase II, 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.
