Wwp2-mediated ubiquitination of the RNA polymerase II large subunit in mouse embryonic pluripotent stem cells.
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.
Li
Hui
H
Key Laboratory of Stem Cell Biology, Institute of Health Sciences, 225 South Chongqing Road, Shanghai 200025, China.
Zhang
Zhihong
Z
Wang
Beibei
B
Zhang
Junmei
J
Zhao
Yingming
Y
Jin
Ying
Y
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000201:enzymology*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D039904:Pluripotent Stem Cells / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
D025801:Ubiquitin; C487995:Wwp2 protein, mouse; D044767:Ubiquitin-Protein Ligases; D012319:RNA Polymerase II; C522173:Rpb1 protein, mouse; D046988:Proteasome Endopeptidase Complex; D008239:Lysine