HECT Domain Ubiquitin-protein Ligase Wwp2 Ubiquitinates The Large Subunit Of RNA Polymerase II

RNA polymerase II is responsible for the synthesis and process of mRNA in eukaryotic cells and is composed of 12 subunits. The largest subunit of RNA polymerase II (Rpb1) plays the most important role, which determinates the initiation and elongation of transcription. Rpb1 has several posttranslational covalent modifications, including phosphorylation, glycosylation and ubiquitination.Previous research has shown that ubiquitination of Rpb1 not only participates in its turnover in normal condition, promotes the degradation of Rpb1 stalled on DNA template during transcription elongation but also promotes the transcription coupled repair when DNA is damaged. Therefore, ubiquitination of Rpb1 enables recovery of RNA synthesis, increases the overall efficiency of transcription, and plays an important role in DNA repair and keeping genome integrity and fidelity. In addition, ubiquitination of Rpb1 may also regulate its activity. Two mammalian E3 ligases of Rpb1 have been reported (pVHL and BRCA1). However, biochemical mechanism underlying its ubiquitination is still elusive.Wwp2 is an E3 ubiquitin-protein ligase previously cloned by our lab as the E3 ligase of transcription factor Oct-4, and is composed of C2 domain, WW domain and HECT (Homologous to E6-AP Carboxyl Terminus,HECT) domain. In the present study, we have found that Wwp2 might interact with Rpb1 through affinity chromatography using GST-WWHECT fusion protein and F9 embryonal carcinoma cell lysate.In this study, we confirmed the specific and phosphorylation independent interaction between Rpb1 and Wwp2 both in vitro and in vivo, which is mediated through WW domain of Wwp2 and CTD(C-terminal domain)of Rpb1, respectively. Importantly, we have found that Wwp2 promotes the ubiquitination of Rpb1 both in vitro and in vivo, and identified the ubiquitination sites in CTD. In addition, the polyubiquitination chain of CTD mediated by Wwp2 is assembled through a new linkage.The ubiquitination and degradation of Rpb1 mediated by Wwp2 is independent on phosphorylation. In normal condition, Wwp2 promotes the ubiquitination and degradation of Rpb1 through ubiquitin proteasome pathway, and controls its steady-state level. The ubiquitination and degradation of hyperphosphorylated Rpb1 mediated by Wwp2 is important for the disassembly of improper transcription complex, enables recovery of RNA synthesis and increases the overall efficiency of transcription. In addition, Wwp2 also participates in the ubiquitination of Rpb1 induced by UV irradiation, which may relate to transcription coupled repair.Our study shows that Wwp2 is a novel HECT domain E3 ubiquitin-protein ligase of Rpb1, and its physiological function is different from the other two known E3 ubiquitin-protein ligases. Therefore, our study on the biochemical mechanism of Wwp2 targeting Rpb1 for ubiquitination will help to reveal the ubiquitination pathway of Rpb1 and its regulation mechanism under physiological and pathological condition.