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In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.

The association of genomic loci to the nuclear periphery is proposed to facilitate cell-type specific gene repression and influence cell fate decisions. However, the interplay between gene position and expression remains incompletely understood, in part because the proteins that position genomic loci at the nuclear periphery remain unidentified. Here, we used an Oligopaint-based HiDRO screen targeting ~1000 genes to discover novel regulators of nuclear architecture in Drosophila cells. We identified the heterochromatin-associated protein, Stonewall (Stwl), as a factor promoting perinuclear chromatin positioning. In female germline stem cells (GSCs), Stwl binds and positions chromatin loci, including GSC differentiation genes, at the nuclear periphery. Strikingly, Stwl-dependent perinuclear positioning is associated with transcriptional repression, highlighting a likely mechanism for the known role of Stwl in GSC maintenance and ovary homeostasis. Thus, our study identifies perinuclear anchors in Drosophila and demonstrates the importance of gene repression at the nuclear periphery for cell fate.Competing Interest StatementThe authors have declared no competing interest.

In most eukaryotic cells, euchromatin is localized in the nuclear interior, whereas heterochromatin is enriched at the nuclear envelope (NE). This conventional chromatin organization is established by heterochromatin tethering to the NE, however its importance for cellular homeostasis is largely unexplored. Peripheral heterochromatin localization relies on redundant NE-tethering systems. One tether is constituted by the lamin B receptor (LBR) in mammals, but the enigmatic nature of the other tethers has hampered functional analyses. Here we demonstrate that the downregulation of abundant, ubiquitous NE proteins can induce the global detachment of heterochromatin from the NE. Among these factors, we identify LBR and LAP2 as major players in bulk heterochromatin attachment to the NE in pluripotent and differentiated mammalian cells. Their loss leads to repositioning of heterochromatin to the nuclear interior, changes in chromatin accessibility, deregulation of gene expression including activation of antiviral innate immunity, and defects in cell fate determination.

DYT1 early-onset dystonia is a severe, incurable disorder of the central nervous system caused by mutations in the gene encoding Torsin1A (Tor1A, DYT1). Torsins are ER-resident AAA+-ATPases implicated in lipid metabolism, nuclear pore complex (NPC) biogenesis, and lipoprotein secretion, yet their molecular function that underlies the disease pathology has remained incompletely understood. Here, we have utilized Drosophila melanogaster and human somatic cells as experimental models to shed light on their mode-of-action. Fly germ cells lacking dTorsin are arrested in development and display defects in the final steps of NPC biogenesis due to a failure in fusion of the inner and outer nuclear membranes. Using proximity labelling of Torsin1A in human cells, we identify the conserved membrane protein chloride channel CLIC-like protein 1 (CLCC1) as a novel Torsin binding partner. Absence of human CLCC1 or its Drosophila homolog dClcc1 phenocopied the membrane fusion defects at NPC assembly sites observed upon Torsin deletion. Furthermore, CLCC1 is enriched at arrested fusion sites, suggesting it to be a candidate for the elusive NE membrane fusogen. Importantly, CLCC1/dClcc1 overexpression is sufficient to rescue NPC biogenesis and developmental defects associated with Torsin-loss-of-function. Taken together, our data suggest that Torsin-regulated CLCC1 activity drives membrane fusion during NPC biogenesis and reveal that modulating CLCC1 expression is a promising therapeutic prospect for DYT1 dystonia.

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.