Delving deeper into RNAPII degradation upon UV-induced DNA damage

INÉS BECKERMAN; JUAN CRISTÓBAL MUÑOZ; MARÍA LUZ REBOTTARO; GONZALO ÁLVAREZ; JOAQUÍN ETCHEPAREBORDA; LEÓN ALBERTO BOUVIER; MANUEL JAVIER MUÑOZ

Buenos Aires

Congreso; Encuentro IFIBYNE Abierto; 2023

IFIBYNE

Among the numerous responses that occur in the event of DNA damage by ultraviolet (UV) light, RPB1, the major and catalytic subunit of human RNA Polymerase II, is specifically degraded by the ubiquitin proteasome system. However, the mechanisms that control RPB1 degradation are not yet fully understood. The “last resort” model proposes that RPB1 is degraded at the damage site, or in cis, in order to facilitate access to the repair machinery. However, RPB1 degradation might not necessarily occur only as a last resort. In this sense, unpublished results from our group indicate that RNAPII degradation is controlled by a DNA damage recognition system which does not rely on the stalling of RNAPII in front of a lesion.Here, we show that, accordingly with an in trans mechanism, UV induced RNAPII degradation is not restricted to active RNAPII molecules. Indeed, the carboxy terminal domain (CTD) of RPB1, which serves as a platform for post-translational modifications that regulate gene expression, is not required for RNAPII degradation. In this sense, two mutant versions of RPB1, one lacking the entire CTD and another one incapable of being phosphorylated on residues which are relevant for transcription, are both degraded upon UV induced DNA damage.On another hand, we have wondered whether it is possible to trigger in trans degradation by transfection of an in vitro damaged plasmid. Unlike exposure of cells with UV light, transfection of in vitro damaged DNA did not trigger RNAPII degradation. Immunofluorescences against cyclobutane pyrimidine dimers (CPDs), the major lesion induced by UV, demonstrated that the damaged plasmid mainly localizes in the cytoplasm. The fact that an in vitro-irradiated plasmid cannot induce RNAPII degradation might be a consequence of differences in the number of nuclear lesions, or a consequence of differences between genomic and plasmidic DNA, such as chromatin structure. While we cannot rule out the former possibility, treatment with trichostatin A (TSA), a histone deacetylase inhibitor that affects chromatin structure, partially inhibited RNAPII degradation.Altogether, these results indicate that RNAPII degradation is not restricted to RPB1 molecules that are engaged in transcription, and that chromatin structure may be involved in the signaling pathway controlling RNAPII levels.