Abstract
RNA polymerase II (RNAPII) acts as a damage sensor for transcription-coupled nucleotide excision repair (TC-NER) and undergoes proteolytic clearance from damaged chromatin by the ubiquitin-proteasome system (UPS). Here, we report that Valosin-containing protein (VCP)/p97, a druggable oncotarget, is essential for RNAPII's proteolytic clearance in mammalian cells. We show that inhibition of VCP/p97, or siRNA-mediated ablation of VCP/p97 and its cofactors UFD1 and UBXD7 severely impairs ultraviolet radiation (UVR)-induced RNAPII degradation. VCP/p97 interacts with RNAPII, and the interaction is enhanced by Cockayne syndrome B protein (CSB). However, the VCP/p97-mediated RNAPII proteolysis occurs independent of CSB. Surprisingly, CSB enhances UVR-induced RNAPII ubiquitination but delays its turnover. Additionally, VCP/p97-mediated RNAPII turnover occurs with and without Von Hippel-Lindau tumor suppressor protein (pVHL), a known substrate receptor of Elongin E3 ubiquitin ligase for RNAPII. Moreover, pVHL re-expression improves cell viability following UVR. Whereas, VCP/p97 inhibition decreases cell viability and enhances a low-dose UVR killing in presence of pVHL. These findings reveal a function of VCP/p97 segregase in UVR-induced RNAPII degradation in mammalian cells, and suggest a role of CSB in coordinating VCP/p97-mediated extraction of ubiquitinated RNAPII and CSB itself from chromatin.
Highlights
Transcription is one of fundamental processes in living cells
To determine whether Valosin-containing protein/p97 (VCP/p97) is involved in UVRinduced degradation of RNA polymerase II (RNAPII) in mammalian cells, we first examined the effect of Valosincontaining protein (VCP)/p97 inhibition on proteolysis of RNAPII by DBeQ, a specific small molecule inhibitor of VCP/p97 ATPase [34]
At 50 J/m2, MG132 and DBeQ prevented RNAPII degradation in ubiquitin specific protease 7 (USP7)-deficient HCT116 cells. These results indicate that VCP/p97 is involved in ubiquitin-mediated RNAPII degradation regardless of the status of USP7
Summary
Transcription is one of fundamental processes in living cells. Transcriptional elongation by RNA polymerase II (RNAPII) often encounters obstacles, including DNA damage, chromatin structures and molecular machineries of other DNA-templated processes, which can cause elongating RNAPII to stall or arrest. The irreversibly arrested RNAPII, e.g., at ultraviolet radiation (UVR)-induced photolesions, can become an obstruction to DNA replication and DNA repair machineries and is highly deleterious to cells. Proteolysis of RNAPII by the ubiquitin-proteasome system (UPS) has been shown to resolve stalled RNAPII [1]. Proteolysis of RNAPII is a tightly regulated multistep process involving ubiquitination of RNAPII, release of the ubiquitin conjugates from chromatin, and proteolytic processing by proteasome. Several E3 ubiquitin ligases including BRCA1-BARD ligase [5, 6], Nedd ligase [7]
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