Abstract Ubiquitination is a post-translational modification responsible for one of the most complex multi-layered communication and regulation system in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Among them, the recently discovered ubiquitin variant Ub KEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. The absence 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. By using data independent acquisition mass spectrometry and microscopy, we show here that despite not impacting protein stability, Ub KEKS is required to maintain normal nucleolar organization. The absence of Ub KEKS increases nucleoli’s size and accentuate their circularity while disrupting dense fibrillar component and fibrillar center structures. Moreover, depletion of Ub KEKS leads to distinct changes in nucleolar composition. Notably, lack of Ub KEKS favors nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis in Ub KEKS knockout cells observed by flow cytometry and real-time cellular growth monitoring. Overall, the results presented here identifies 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 already complex ubiquitination system.

CRISPR/Cas9-mediated knockout of the ubiquitin variant UbKEKSreveals a role in regulating nucleolar structures and composition