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The nuclear pore complex (NPC) regulates transport of macromolecules into and out of the nucleus. A study now shows that mechanical force applied on the nucleus affects the transport rates across the NPC diffusion barrier, modulating the nuclear localization of certain cargos.

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. Ribosomes are the molecular machines in cells that produce proteins. The ribosomes themselves are composed of almost 80 different proteins that are held together by scaffolds made from molecules of RNA. Each protein is present in one copy, and so equal numbers of all proteins are needed to assemble a ribosome. However, because it takes many steps to produce a protein and biological processes are inherently imprecise, it is essentially impossible for a cell to produce exactly the same number of copies of all the proteins in a ribosome. Much research suggests that, to overcome these issues, a cell will make more of certain ribosomal proteins than it needs, and then degrade the leftovers that are not used. However, it was not clear how this happens, nor was it known what are the consequences of failing to degrade the leftovers. Now, Sung et al. show that yeast cells use an enzyme named Tom1 to attach a protein-marker called ubiquitin to ribosomal proteins that are made in excess and not assembled into ribosomes. The ubiquitin serves as a tag that marks proteins for degradation, and yeast cell that lack Tom1 fail to degrade any excess ribosomal proteins. Consequently, the mutant yeast become sensitive to any factors that alter the balance of the protein and RNA building blocks used to assemble ribosomes. The human equivalent of Tom1 is known as Huwe1, and the data of Sung et al. suggest that this enzyme acts in a similar pathway. Further experiments are now needed to explore the role of Huwe1 in greater depth, and investigate if problems with this enzyme are associated with any human diseases. Finally, working out the exactly how Tom1 recognizes unassembled ribosomal proteins will be another important challenge for future studies.

Nuclear pore complexes (NPCs) consist of around 1000 protein subunits, are embedded in the membrane that surrounds the nucleus, and regulate transport between the nucleus and the cytoplasm. Although the overall shape of NPCs is known, the details of this macromolecular complex have been obscure. Now, Lin et al. have reconstituted the pore components, determined the interactions between them, and fitted them into a tomographic reconstruction. Kosinski et al. have provided an architectural map of the inner ring of the pore. Science , this issue pp. 10.1126/science.aaf1015 and 363 Reconstitution, spectroscopy, and crystallography allow the construction of a model of the human nuclear pore. The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC’s structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.

This Perspective discusses how two complementary approaches, bottom-up in vitro and top-down in situ structural biology, have now converged to generate the first predictive structural models of the nuclear pore scaffold. Elucidating the structure of the nuclear pore complex (NPC) is a prerequisite for understanding the molecular mechanism of nucleocytoplasmic transport. However, owing to its sheer size and flexibility, the NPC is unapproachable by classical structure determination techniques and requires a joint effort of complementary methods. Whereas bottom-up approaches rely on biochemical interaction studies and crystal-structure determination of NPC components, top-down approaches attempt to determine the structure of the intact NPC in situ . Recently, both approaches have converged, thereby bridging the resolution gap from the higher-order scaffold structure to near-atomic resolution and opening the door for structure-guided experimental interrogations of NPC function.

Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery. Acl4 is a dedicated assembly chaperone for ribosomal protein RpL4 that recognizes RpL4 in the cytoplasm to facilitate its nuclear import. Here the authors reveal the mechanism whereby Acl4 recognizes RpL4 and functions to protect it from Tom1-mediated degradation until RpL4 incorporation into the maturing 60S pre-ribosomal subunit.

The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP 6 , unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1•Nup42•DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans. The export of mRNA to the cytosol depends on the nuclear pore complex (NPC) and the activation of the helicase DDX19, but their interplay in humans remains poorly understood. Here, the authors present a structural and functional analysis of DDX19 activation, revealing how the human NPC regulates mRNA export.

The reversible methylation of specific lysine residues in histone tails is crucial in epigenetic gene regulation. LSD1, the first known lysine-specific demethylase, selectively removes monomethyl and dimethyl, but not trimethyl modifications of Lys4 or Lys9 of histone-3. Here, we present the crystal structure of LSD1 at 2.9-Å resolution. LSD1 forms a highly asymmetric, closely packed domain structure from which a long helical 'tower' domain protrudes. The active site cavity is spacious enough to accommodate several residues of the histone tail substrate, but does not appear capable of recognizing the different methylation states of the substrate lysine. This supports the hypothesis that trimethylated lysine is chemically rather than sterically discriminated. We present a biochemical analysis of LSD1 mutants that identifies crucial residues in the active site cavity and shows the importance of the SWIRM and tower domains for catalysis.

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here we present the crystal structure of a reconstituted ∼400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central \"triskelion\" of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ∼16 megadalton of its mass.

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ∼425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.

The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 Å resolution. Rae1 forms a seven-bladed β-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an [almost equal to]50-Å-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 β-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1{bullet}Nup98GLEBS surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1{bullet}Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.