Wwp2-mediated ubiquitination of the RNA polymerase II large subunit in mouse embryonic pluripotent stem cells
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Updated: Jul 14, 2026
Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events
Published on: May 13, 2019
Hui Li1, Zhihong Zhang, Beibei Wang
1Key Laboratory of Stem Cell Biology, Institute of Health Sciences, 225 South Chongqing Road, Shanghai 200025, China.
Abstract:
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.
Insights
Wwp2, a novel ubiquitin ligase, targets the large subunit of RNA polymerase II (Rpb1) for degradation independently of DNA damage. This identifies Wwp2
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Ubiquitination and degradation of RNA polymerase II large subunit (Rpb1) are crucial for DNA damage response and transcription regulation.
- The specific ubiquitin ligases mediating DNA damage-independent Rpb1 regulation in mammals were previously unknown.
Purpose of the Study:
- To identify novel ubiquitin ligases involved in DNA damage-independent regulation of Rpb1 in mammalian cells.
- To characterize the interaction and ubiquitination mechanism of Wwp2 on Rpb1.
Main Methods:
- Co-immunoprecipitation assays to assess protein interactions.
- In vitro and in vivo ubiquitination assays.
- Western blotting to analyze protein levels and ubiquitination status.
- Site-directed mutagenesis to identify ubiquitination sites.
Main Results:
- Wwp2, a HECT domain ubiquitin E3 ligase, was identified as a novel Rpb1-interacting protein.
- Wwp2 specifically ubiquitinated Rpb1 in a manner independent of Rpb1 phosphorylation or DNA damage.
- The interaction involved Wwp2's WW domain and Rpb1's C-terminal domain.
- Downregulation of Wwp2 led to reduced Rpb1 ubiquitination and increased Rpb1 protein levels.
- Six lysine residues in Rpb1's C-terminal domain were identified as Wwp2-mediated ubiquitination sites.
Conclusions:
- Wwp2 is a key ubiquitin ligase regulating Rpb1 levels under normal physiological conditions.
- Wwp2-mediated ubiquitination of Rpb1 contributes to the control of Rpb1 protein homeostasis.
- This study reveals a novel mechanism for regulating transcription by controlling Rpb1 stability.
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