Delving deeper into RNAPII degradation upon UV-induced DNA damage

INÉS BECKERMAN; JUAN CRISTÓBAL MUÑOZ; LEÓN ALBERTO BOUVIER; MANUEL JAVIER MUÑOZ

Colonia

Congreso; Primer Encuentro Binacional de los Clubes del RNA de Argentina y Uruguay; 2022

Clubes del RNA de Argentina y Uruguay

DNAis the only biopolymer that is neither disposable nor recyclable so it must be repaired when damaged. Among the various repair systems that human cells have, the Nucleotide Excision Repair system is the most relevant for repair of ultraviolet 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 actively transcribed genes, as recognition is accomplished by an RNA Polymerase II that cannot bypass the insult and therefore gets stalled. Both detection systems then converge on the machinery that actually repairs the damage.RPB1, 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 RPB1 degradation are not yet fully understood The “last resort” model proposes that the arrest of RNAPII in DNA given its inability to transcribe the lesions would lead to its degradation, thus allowing access to the repair machinery. Thus, according to this model, RPB1 is degraded in cis throughTC-NER. However, RPB1 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 KO cells). RNAPII degradation in this GG-NER KO cell line was markedly inhibited, indicating that damage recognition through GG NER also controls RNAPII levels Accordingly with an in trans mechanism, UV induced RNAPII degradation is notrestricted to phosphorylated molecules, and an RPB 1 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 one hand, treatment with valproic acid, a histone deacetylase inhibitor, enhanced RNAPII degradation. On the other hand, transfection of a naked, UV damaged plasmid did not elicit RNAPII degradation.Having in mind that it has been recently demonstrated that RNAPII levels shape the gene Expression response upon UV irradiation, understanding the mechanisms that govern its degradation is of paramount importance.