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

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 UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. The absence of UbKEKS 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, UbKEKS is required to maintain normal nucleolar organization. The absence of UbKEKS increases nucleolis size and accentuate their circularity while disrupting dense fibrillar component and fibrillar center structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Notably, lack of UbKEKS favors nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis in UbKEKS knockout cells observed by flow cytometry and real-time cellular growth monitoring. Overall, the results presented here identifies the first cellular functions of the UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the already complex ubiquitination system.