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 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 UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. Depletion 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. Using data-independent acquisition mass spectrometry and microscopy, we show that despite not impacting protein stability, UbKEKS is required to maintain a normal nucleolar organization. The absence of UbKEKS increases nucleoli's size and accentuate their circularity while disrupting dense fibrillar component and fibrillar centre structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Lack of UbKEKS 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 UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the ubiquitination system.

Culturable Bacterial Diversity from the Basaltic Subsurface of the Young Volcanic Island of Surtsey, Iceland.

The oceanic crust is the world's largest and least explored biosphere on Earth. The basaltic subsurface of Surtsey island in Iceland represents an analog of the warm and newly formed-oceanic crust and offers a great opportunity for discovering novel microorganisms. In this study, we collected borehole fluids, drill cores, and fumarole samples to evaluate the culturable bacterial diversity from the subsurface of the island. Enrichment cultures were performed using different conditions, media and temperatures. A total of 195 bacterial isolates were successfully cultivated, purified, and identified based on MALDI-TOF MS analysis and by 16S rRNA gene sequencing. Six different clades belonging to Firmicutes (40%), Gammaproteobacteria (28.7%), Actinobacteriota (22%), Bacteroidota (4.1%), Alphaproteobacteria (3%), and Deinococcota (2%) were identified. Bacillus (13.3%) was the major genus, followed by Geobacillus (12.33%), Enterobacter (9.23%), Pseudomonas (6.15%), and Halomonas (5.64%). More than 13% of the cultured strains potentially represent novel species based on partial 16S rRNA gene sequences. Phylogenetic analyses revealed that the isolated strains were closely related to species previously detected in soil, seawater, and hydrothermal active sites. The 16S rRNA gene sequences of the strains were aligned against Amplicon Sequence Variants (ASVs) from the previously published 16S rRNA gene amplicon sequence datasets obtained from the same samples. Compared with the culture-independent community composition, only 5 out of 49 phyla were cultivated. However, those five phyla accounted for more than 80% of the ASVs. Only 121 out of a total of 5642 distinct ASVs were culturable (≥98.65% sequence similarity), representing less than 2.15% of the ASVs detected in the amplicon dataset. Here, we support that the subsurface of Surtsey volcano hosts diverse and active microbial communities and that both culture-dependent and -independent methods are essential to improving our insight into such an extreme and complex volcanic environment.

UBB pseudogene 4 encodes functional ubiquitin variants.

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.