Cellular consequences of RNA damage and its resolution

Cordes, Jacqueline (2025): Cellular consequences of RNA damage and its resolution. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

RNA is an essential cellular molecule and exerts many different functions to maintain homeostasis. It not only plays a critical part in the central dogma, where mRNA directs protein synthesis, but also has many other regulatory and catalytic functions. Therefore, it is important to maintain the integrity of RNA in cells. Due to its presumably transient lifetime, damage to RNA has long been disregarded, but increasing evidence shows that RNA is easily damaged by endogenous and environmental agents, and that such RNA damage can have detrimental consequences to cells. Studying the specific response to RNA damage is difficult, as many damaging agents act pleiotropically and therefore cause confounding DNA damage, inducing a complex damage response. To overcome this problem, we aim to use nucleoside analogues that specifically incorporate into and thereby damage RNA. Hence, this will allow us to study the cellular stress response to and resolution mechanisms of RNA damage and will help to uncover the RNA component of nucleic acid damage. In the first study, we hypothesize that endogenous formaldehyde, which is produced in substantial quantities in cells, can cause damage by crosslinking proteins to RNA. To study mRNA-protein crosslinks (mRPCs), we employed photoactivatable nucleoside-enhanced crosslinking (PAR-CL), more specifically incorporation of nucleoside analogue 4-SU into cellular RNA, followed by UVA irradiation. We show that mRPCs are toxic to cells and lead to severe translation defects. Furthermore, the integrated stress response (ISR), the ribotoxic stress response (RSR), as well as ribosome-associated quality control (RQC) are triggered by mRPCs. Importantly, cells can recover from sub-lethal doses of mRPC induction and we identify a translation-coupled resolution mechanism, which targets mRPCs for proteasomal degradation. The detection and resolution of mRPCs is dependent on ribosome collisions and involves the ribosome collision sensor GCN1 and the ubiquitin E3 ligases RNF14 and RNF25. In detail, RNF14 modifies mRPCs with K48- and K6-linked ubiquitin chains in a translation- dependent manner. This is supported by GCN1 and RNF25 and results in degradation of protein adduct by the proteasome. In a second study, we aim to characterize the molecular basis for the mechanism of action of the chemotherapeutic drug azacytidine (aza-C). aza-C is a cytidine analogue and is known to be incorporated into DNA and RNA. In contrast, the chemically similar drug decitabine (aza-dC) is only incorporated into DNA, but both compounds are used in an interchangeable manner in treatment of acute myeloid leukaemia (AML). To understand the cellular consequences of aza-C incorporation into RNA, we performed genome-wide CRISPR screens, comparing aza-C and aza-dC treatment. We show that tolerance to aza-C is mediated by ribosome-associated E3 ligase RNF25. Furthermore, aza-C treatment induces translation 8 defects and strong activation of the integrated stress response (ISR), resulting in cell death. More precisely, aza-C incorporation into mRNA causes ribosome stalling and collisions, likely due to obstruction of the ribosomal A-site by spontaneous ring-opening of incorporated aza-C. Ribosome collisions then engage GCN1 and RNF25 subsequently ubiquitylates ribosomal proteins in the 40S and 60S subunit. We believe these modifications blunt activation of the ISR by GCN2 and therefore provide tolerance to aza-C. These studies not only emphasize RNA damage and its cellular consequences as an important component of nucleic acid damage, but also allow characterization of the response to physiologically relevant RNA lesions. Thus, these insights provide a basis to clarify the role of RNA damage in disease development and could improve the use of RNA-damaging agents as tool in cancer treatment.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: RNA damage, Nucleic acid damage, Cellular quality control, RNA-protein crosslinks, Formaldehyde, UV, Translation, Ubiquitylation, Azacytidine, Acute myeloid leukemia
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 1. July 2025
• 1. Referee: Stingele, Julian
• Signature of the printed copy: 0001/UMC 32085