Studio di nuove funzioni di TSPYL2 nella regolazione della struttura nucleolare e nella risposta allo stress ribosomiale

The nucleolus is a nuclear compartment best known for its role in ribosome biogenesis, but it also acts as a central hub in the coordination of cellular stress responses. Perturbations in its structure or activity, such as inhibition of rRNA transcription, trigger the nucleolar stress response, leading to activation of the tumor suppressor p53. Despite increasing evidence linking nucleolar integrity to p53 regulation, the molecular mechanisms coordinating these processes remain incompletely understood. Testis Specific Protein Y-encoded-like 2 (TSPYL2) is a protein of the Nucleosome Assembly Protein (NAP) superfamily that has been implicated in cell cycle control and DNA damage response, where it enhances p53 acetylation through inhibition of SIRT1 and activation of p300. Moreover, TSPYL2 has been reported to localize to the nucleolus, suggesting a possible role in linking nucleolar organization to p53 activation. In this project, I investigated the hypothesis that TSPYL2 participates in the maintenance of nucleolar architecture and in the regulation of p53 activity during nucleolar stress. A proteomic screening of TSPYL2-associated proteins revealed an enrichment of nucleolar and ribosome biogenesis factors, supporting the idea that TSPYL2 acts within the nucleolar environment. Among these interactors, the helicase DDX24, previously linked to nucleolar structure and p53 regulation, was identified as a potential mediator of TSPYL2 function. Functional analyses demonstrated that loss of TSPYL2 profoundly alters nucleolar organization. Cells depleted of TSPYL2 displayed enlarged and irregularly shaped nucleoli, accompanied by increased transcription of the 47S precursor rRNA, indicating an upregulation in rRNA synthesis. Importantly, TSPYL2 depletion also resulted in reduced p53 acetylation, demonstrating that TSPYL2 is necessary for the full activation of p53 in response to nucleolar stress. In contrast, DDX24 depletion increased p53 acetylation and total protein levels, and double silencing experiments placed TSPYL2 downstream of DDX24 in this regulatory pathway. Mechanistically, TSPYL2 and DDX24 were found to compete for binding to the acetyltransferase p300, further consolidating their role in p53 regulation. Together, these findings indicate that TSPYL2 is a critical regulator of nucleolar homeostasis and a positive modulator of p53 acetylation. By coordinating the structural and functional state of the nucleolus with the cellular stress response, TSPYL2 may serve as the missing molecular link between these two processes.