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The nuclear envelope (NE) is connected to the different types of cytoskeletal elements by linker of nucleoskeleton and cytoskeleton (LINC) complexes. LINC complexes exist from yeast to humans, and have preserved their general architecture throughout evolution. They are composed of SUN and KASH domain proteins of the inner and the outer nuclear membrane, respectively. These SUN–KASH bridges are used for the transmission of forces across the NE and support diverse biological processes. Here, we review the function of SUN and KASH domain proteins in various unicellular and multicellular species. Specifically, we discuss their influence on nuclear morphology and cytoskeletal organization. Further, emphasis is given on the role of LINC complexes in nuclear anchorage and migration as well as in genome organization.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins 1 , 2 . Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1 3 . Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO1 4 . The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae . Studies of five cryo-electron microscopy structures reveal the composition and conformational progression in the final maturation events of human 40S ribosomal subunit assembly.

Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s −1 in vivo. Ribosomal biogenesis is known to require nuclear to cytoplasmic export, but the precise kinetics remain unclear. Here, the authors use super-resolution confocal microscopy and single molecule tracking to visualize export of single pre-60S particles through nuclear pore complexes.

Key Points The organization of the nuclear envelope (NE) is perfectly adapted to its function as a compartment boundary and protective coat for the genome. The plasticity of the NE allows it to withstand considerable mechanical challenges, whether cells are embedded within tissues or during migration. In the case of NE rupture, membrane lesions are rapidly repaired to avoid genome damage. Local NE remodelling is required for the biogenesis of nuclear pore complexes, the nuclear egress of large particles such as ribonucleoprotein complexes or certain viruses and the elimination of defective NE components. Distinct modes of NE re-organization evolved for the process of nuclear division, of which the complete disassembly and reassembly of the NE during open mitosis represents an extreme case. Changes in NE composition and morphology accompany differentiation and contribute to tissue-specific cell function. The nuclear envelope is more than a static barrier between the nuclear and cytoplasmic compartments. It is very dynamic and undergoes extensive remodelling in response to mechanical challenges as well as during cell division, growth and differentiation. As a compartment border, the nuclear envelope (NE) needs to serve as both a protective membrane shell for the genome and a versatile communication interface between the nucleus and the cytoplasm. Despite its important structural role in sheltering the genome, the NE is a dynamic and highly adaptable boundary that changes composition during differentiation, deforms in response to mechanical challenges, can be repaired upon rupture and even rapidly disassembles and reforms during open mitosis. NE remodelling is fundamentally involved in cell growth, division and differentiation, and if perturbed can lead to devastating diseases such as muscular dystrophies or premature ageing.

Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

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.

Background Ribosomes are huge ribonucleoprotein particles that mediate protein synthesis in all organisms. The synthesis of ribosomes is a complex process that involves hundreds of supporting factors in mammalian cells, including proto-oncogenes and tumor suppressors. Dysregulation of ribosome biogenesis can contribute to tumorigenesis, and the increased production of ribosomes in cancer cells is known to promote proliferative cell growth. Therefore, ribosome biogenesis represents an attractive vulnerability of cancer cells that ought to be exploited for the development of anti-cancer drugs. Despite the large number of trans-acting factors promoting ribosome assembly including potentially druggable enzymes, only few chemical inhibitors that act on ribosome biogenesis, especially downstream of pre-rRNA transcription, have been identified to date. Results To enable large-scale screens for chemical compounds that interfere with ribosome biogenesis, we have established a pipeline to perform single-cell, imaging-based screening campaigns using four different readouts, including fluorescent ribosomal protein reporters (RPS2-YFP, RPL29-GFP) and immunofluorescence analyses of the ribosome biogenesis factor ENP1(BYSL), in HeLa cells, a human cancer line. We have assessed the robustness of our high-content screening approach by performing a pilot screen using a library comprising more than 1000 FDA-approved drugs with known targets in other pathways. This pilot screen obtained excellent quality scores and identified ten compounds as hits. These hit compounds likely affect ribosome synthesis indirectly, the majority by inducing DNA damage or by inhibiting the proteasome. We therefore used the identified compounds to establish appropriate counter assays for DNA damage and proteasome inhibition, to exclude common indirect effects in the downstream analysis of such screening campaigns. Conclusions The established screening pipelines provide a robust, efficient, and sensitive experimental framework to identify chemical compounds that impair ribosome synthesis. The combination of readouts allows to distinguish effects on pre-rRNA synthesis from downstream effects on ribosome assembly. Established counter assays on DNA damage and protein degradation enable to exclude effects on these pathways, which commonly interfere with ribosome synthesis indirectly. The developed assays are easily scalable to screen libraries of higher complexity in the future.

Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent. Exploiting in vitro reconstitution and in vivo analysis of INM targeting, we establish that Atlastins, membrane-bound GTPases of the ER, sustain the efficient targeting of proteins to the INM by their continued activity in preserving ER topology. When ER topology is altered, the long-range diffusional exchange of proteins in the ER network and targeting efficiency to the INM are diminished. Highlighting the general importance of proper ER topology, we show that Atlastins also influence NPC biogenesis and timely exit of secretory cargo from the ER.

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.