Delving deeper into RNAPII degradation upon UV-induced DNA damage

INES BECKERMAN; JUAN CRISTOBAL MUÑOZ; LEON ALBERTO BOUVIER; MANUEL JAVIER MUÑOZ

Colonia

Congreso; 1st Binational Congress of RNA Clubs in Argentina and Uruguay, December 2nd and 3rd, 2022, Colonia, Uruguay; 2022

RNA Clubs from Argentina and Uruguay

DNA is the only blapolymer that is nelther dísposable nor recyclable so lt must be repalred when damaged. Among the various repair systems that human cells have, the Nucleotlde Exclsion Repair (NER) system is the most relevant for repair of ultraviolet (UV) light•induced DNA lesions. Damage detection by NER system occurs by two different lesion·sensing mechanisms: Global Genome repair (GG-NER), which relies on factors XPE and XPC, which detect damage throughout the genome, and Transcription-Coupled repair (TC-NER), which detects damage exclusively on the template strands of actlvely transcribed genes, as recognition is accomplished by an RNA Polymerase 11 (RNAPII) that cannot bypass the insult and therefore gets stalled. Both detection systems then converge on the machlnery that actually repairs the damage. RPBl, the major and catalytic subunit of human RNAPII, is specifically degraded by the ubiquitin-proteasome system upon UV-induced DNA damage. However, the mechanisms that control RPBl degradation are not yet fully understood. The "last resort• model proposes that the arrest of RNAPII in DNA -given its inabllity to transcribe the lesions-would lead to its degradation, thus allowing access to the repair machinery. Thus, accordlng to thls model, RPBl Is degraded in cis, through TC-NER. However, RPBl degradation might not necessarily occur only as a last resort to give access to the repair machinery. Here, we propose an altogether different mechanism that regulates RNAPII degradation. Using CRISPR/Cas9, we generated human keratinocytes unable to globally recognize lesions through the GG•NER system (XPC & XPE double KOcells). RNAPII degradation in this GG-NER KO cell line was markedly inhlbited, indicating that damage recognition through GG-NER also controls RNAPII levels. Accordingly with an in trans mechanism, UV-induced RNAPII degradatlon Is no restricted to phosphorylated molecules, and an RPBl mutant which is incapable of transcribing can also be degraded upon UV. Also in agreement with this model, degradation at late time points after UV-irradiation is not dependent on the ubiquitin-dependent segregase VCP/p97, which suggests that this process does not imply eviction of RNAPII from chromatin. Finally, preliminary results show that chromatin structure influences RNAPII degradation: on the ene hand, treatment with valproic acid, a histone deacetylase inhibitor, enhanced RNAPII degradation. On the other hand, transfection f a naked, UV-damaged plasmld did not elicit RNAPII degradation. Having in mind that it has been recently demonstrated that RNAPII levels shape the gene expression response upen UV-rradiation, understanding the mechanisms that govern its degradation is of paramount importance.