CRISPR/Cas9-mediated knockout of the ubiquitin variant Ub(KEKS) reveals a role in regulating nucleolar structures and composition

Ubiquitination is a post-translational modification responsible for one of the most complex multilayered communication and regulation systems in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Recently discovered ubiquitin variant Ub(KEKS) can specifically target several proteins and yet, functional consequences of this new modification remain unknown. Depletion of Ub(KEKS) induces accumulation of lamin A in the nucleoli, highlighting the need for deeper investigations about protein composition and functions regulation of this highly dynamic and membrane-less compartment. Using data-independent acquisition mass spectrometry and microscopy, we show that despite not impacting protein stability, Ub(KEKS) is required to maintain a normal nucleolar organization. The absence of Ub(KEKS) increases nucleoli's size and accentuate their circularity while disrupting dense fibrillar component and fibrillar centre structures. Moreover, depletion of Ub(KEKS) leads to distinct changes in nucleolar composition. Lack of Ub(KEKS) favours nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis observed by flow cytometry and real-time monitoring. Overall, these results identify the first cellular functions of the Ub(KEKS) variant and lay the foundation stone to establish Ub(KEKS) as a new universal layer of regulation in the ubiquitination system.