CSA protein plays a key role in transcription-coupled nucleotide excision repair (TC-NER), a molecular pathway dedicated to the removal of UV-induced DNA lesions that block the progression of RNA polymerase II transcription. Mutations in the ERCC8/CSA gene cause Cockayne syndrome type A (CS-A), a rare autosomal recessive multisystem disorder characterised by physical and mental delay, cutaneous photosensitivity and signs of premature ageing, including progressive neurodegeneration. More recent studies have identified additional roles for CSA beyond TC-NER; in particular, CSA has been implicated in the cellular response to oxidative stress, mitochondrial functionality, RNA polymerase I- and II-mediated transcription, nucleolar stress response and protein synthesis infidelity. However, the precise mechanisms and extent of CSA involvement in many of these pathways remain poorly understood. To further investigate the role of CSA in these processes, our laboratory generated specific isogenic cell lines expressing at physiological levels either the wild-type or mutated forms of recombinant CSA. For this work of thesis, the isogenic cell lines have been used to further investigate the molecular mechanisms underlying CS pathological condition. In detail, immunoblot analysis has been used to evaluate p21 and p53 protein levels, two markers of nucleolar stress. In addition, the levels of RPS3 and RPS10 ribosomal proteins have been analysed. RPS3 and RPS10 are novel CSA-interacting proteins identified in the host laboratory as co-factors of RNA polymerase I in ribosomal transcription. These CSA-interactors are well-known key players in ribosome quality control (RQC), a mechanism that is activated upon ribosome collision and translation stalling. Compared to cells expressing the wild-type CSA, we found a significant accumulation of p21 and p53 protein levels in all cell lines carrying mutated CSA, indicative of nucleolar stress. In addition, we also observed increased levels of RPS10 in these cells, whereas no clear changes of RPS3 were found. Since RPS3 and RPS10 become targets of the ubiquitination process upon RQC activation, we used the UBI-QAPTURE-Q immunoprecipitation technique to isolate the ubiquitinated proteins present within cell extracts. This approach revealed increased levels of mono-ubiquitinated RPS10 in all the mutant cell lines compared to the control wild-type CSA. Furthermore, besides the ubiquitination, additional post-translational modifications were found in both RPS3 and RPS10. These modifications were lost in the isogenic cell lines carrying the mutated forms of CSA, demonstrating that they require the presence of a functional CSA protein. Next, we extended our analyses to primary dermal fibroblasts isolated from patients with CS clinical features and biallelic mutations in ERCC8/CSA gene. Differently from the isogenic cell lines, no clear changes in p21 or p53 protein amount were detected in primary cells, whereas increased levels of RPS3 were found in CS4PV and CS7PV patients’ cells. The differences observed between primary and isogenic cell lines might be caused by their different proliferation rates, as the isogenic lines were derived from cells immortalized with the Simian Virus 40 (SV40) large T antigen. Instead, similarly to the isogenic cell lines, the presence of additional post-translational modifications besides ubiquitination was found on RPS10, demonstrating that these events directly or indirectly depend on CSA functionality. Overall, this thesis provides new insights into the role of CSA in nucleolar metabolism and its impact on the ubiquitination profiles of specific ribosomal proteins. They represent a starting point for further investigations that may uncover the molecular link between ribosomal transcription, ribosome collisions and loss of proteostasis in patients with Cockayne syndrome.
La proteina CSA svolge un ruolo chiave nel meccanismo di riparazione del DNA TC-NER, una via di segnale dedicata alla rimozione delle lesioni del DNA causate dai raggi UV, che bloccano la progressione dell’RNApol II. Mutazioni nel gene CSA possono causare l’insorgenza della sindrome di Cockayne di tipo A, una rara malattia multisistemica autosomica recessiva, caratterizzata da ritardo dello sviluppo, fotosensibilità cutanea, tratti progeroidi tra cui neurodegenerazione. Studi recenti hanno identificato ulteriori ruoli di CSA al di fuori del TC-NER in particolare la proteina è coinvolta nella risposta cellulare allo stress ossidativo, nella funzione mitocondriale, nella trascrizione mediata dalle RNApol I e II, e nella risposta allo stress nucleolare. Tuttavia, i meccanismi esatti e l’entità del coinvolgimento della proteina CSA sono ad oggi ancora poco chiari. Per approfondire il ruolo di CSA in questi processi, il nostro laboratorio ha generato specifiche linee cellulari isogeniche esprimenti a livelli fisiologici, la forma wild-type o forme mutate di CSA ricombinante che sono state utilizzate per approfondire i meccanismi molecolari alla base della condizione patologica CS. In particolare, sono stati valutati i livelli delle proteine p21 e p53, due marcatori di stress nucleolare. Inoltre, sono stati analizzati i livelli delle proteine ribosomiali RPS3 e RPS10 che il laboratorio ospitante ha recentemente identificato come cofattori della RNA pol I in un complesso multiproteico contenente anche CSA. Questi nuovi interattori di CSA sono regolatori chiave del controllo di qualità dei ribosomi, un meccanismo che viene attivato in seguito a collisione dei ribosomi e conseguente blocco della traduzione. Rispetto alle cellule che esprimono la forma wtCSA, abbiamo riscontrato un significativo accumulo dei livelli proteici di p21 e p53 in tutte le linee cellulari portatrici delle forme mutate, indicativo di stress nucleolare. Inoltre, abbiamo osservato un aumento dei livelli di RPS10 in queste cellule, mentre non sono state rilevate variazioni di RPS3. Poiché RPS3 e RPS10 sono conosciute per essere ubiquitinate in seguito alla collisione dei ribosomi, abbiamo utilizzato la tecnica di immunoprecipitazione UBI-QAPTURE-Q per isolare le proteine cellulari ubiquitinate. Questo approccio ha evidenziato un aumento dei livelli di RPS10 monoubiquitinata in tutte le linee cellulari mutate rispetto al controllo. Oltre all’ubiquitinazione, sono state rilevate ulteriori modificazioni post-traduzionali sia a carico di RPS3 che di RPS10. Tali modificazioni sono assenti nelle linee cellulari isogeniche portatrici delle forme mutate di CSA, dimostrando che la loro presenza richiede la proteina wild-type. Successivamente, abbiamo esteso le nostre analisi a fibroblasti primari isolati dal derma di pazienti CS e portatori di mutazioni bialleliche nel gene CSA. A differenza delle linee cellulari isogeniche, nelle cellule primarie non sono state rilevate variazioni evidenti nei livelli proteici di p21 o p53, mentre è stato osservato un aumento dei livelli di RPS3 nelle cellule dei pazienti. Le differenze tra le linee cellulari isogeniche e quelle primarie potrebbero derivare dalla diversa capacità proliferativa, poiché le linee isogeniche derivano da cellule immortalizzate con SV40. Analogamente a quanto osservato nelle linee cellulari isogeniche, oltre all’ubiquitinazione è stata rilevata la presenza di ulteriori modificazioni di RPS10, il che dimostra che tali eventi dipendono, direttamente o indirettamente, dalla funzionalità di CSA. Nel complesso, questa tesi fornisce nuove conoscenze sul ruolo di CSA nel metabolismo nucleolare e il suo impatto sull’ubiquitinazione di specifiche proteine ribosomali. I risultati ottenuti rappresentano un punto di partenza per ulteriori studi volti a chiarire il legame molecolare tra la trascrizione ribosomiale, le collisioni dei ribosomi e la perdita di proteostasi nei pazienti affetti da CS.
Nuove prospettive sullo stress nucleolare nella sindrome di Cockayne di tipo A
RUŇANINOVÁ, EMA
2025/2026
Abstract
CSA protein plays a key role in transcription-coupled nucleotide excision repair (TC-NER), a molecular pathway dedicated to the removal of UV-induced DNA lesions that block the progression of RNA polymerase II transcription. Mutations in the ERCC8/CSA gene cause Cockayne syndrome type A (CS-A), a rare autosomal recessive multisystem disorder characterised by physical and mental delay, cutaneous photosensitivity and signs of premature ageing, including progressive neurodegeneration. More recent studies have identified additional roles for CSA beyond TC-NER; in particular, CSA has been implicated in the cellular response to oxidative stress, mitochondrial functionality, RNA polymerase I- and II-mediated transcription, nucleolar stress response and protein synthesis infidelity. However, the precise mechanisms and extent of CSA involvement in many of these pathways remain poorly understood. To further investigate the role of CSA in these processes, our laboratory generated specific isogenic cell lines expressing at physiological levels either the wild-type or mutated forms of recombinant CSA. For this work of thesis, the isogenic cell lines have been used to further investigate the molecular mechanisms underlying CS pathological condition. In detail, immunoblot analysis has been used to evaluate p21 and p53 protein levels, two markers of nucleolar stress. In addition, the levels of RPS3 and RPS10 ribosomal proteins have been analysed. RPS3 and RPS10 are novel CSA-interacting proteins identified in the host laboratory as co-factors of RNA polymerase I in ribosomal transcription. These CSA-interactors are well-known key players in ribosome quality control (RQC), a mechanism that is activated upon ribosome collision and translation stalling. Compared to cells expressing the wild-type CSA, we found a significant accumulation of p21 and p53 protein levels in all cell lines carrying mutated CSA, indicative of nucleolar stress. In addition, we also observed increased levels of RPS10 in these cells, whereas no clear changes of RPS3 were found. Since RPS3 and RPS10 become targets of the ubiquitination process upon RQC activation, we used the UBI-QAPTURE-Q immunoprecipitation technique to isolate the ubiquitinated proteins present within cell extracts. This approach revealed increased levels of mono-ubiquitinated RPS10 in all the mutant cell lines compared to the control wild-type CSA. Furthermore, besides the ubiquitination, additional post-translational modifications were found in both RPS3 and RPS10. These modifications were lost in the isogenic cell lines carrying the mutated forms of CSA, demonstrating that they require the presence of a functional CSA protein. Next, we extended our analyses to primary dermal fibroblasts isolated from patients with CS clinical features and biallelic mutations in ERCC8/CSA gene. Differently from the isogenic cell lines, no clear changes in p21 or p53 protein amount were detected in primary cells, whereas increased levels of RPS3 were found in CS4PV and CS7PV patients’ cells. The differences observed between primary and isogenic cell lines might be caused by their different proliferation rates, as the isogenic lines were derived from cells immortalized with the Simian Virus 40 (SV40) large T antigen. Instead, similarly to the isogenic cell lines, the presence of additional post-translational modifications besides ubiquitination was found on RPS10, demonstrating that these events directly or indirectly depend on CSA functionality. Overall, this thesis provides new insights into the role of CSA in nucleolar metabolism and its impact on the ubiquitination profiles of specific ribosomal proteins. They represent a starting point for further investigations that may uncover the molecular link between ribosomal transcription, ribosome collisions and loss of proteostasis in patients with Cockayne syndrome.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/37405