NUP98 and RAE1 sustain progenitor function through HDAC-dependent chromatin targeting to escape from nucleolar localization.

Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors' regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98's chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98's chromatin binding and dysregulates NUP98 and RAE1's target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation.

Matrin 3 in neuromuscular disease: physiology and pathophysiology.

RNA-binding proteins (RBPs) are essential factors required for the physiological function of neurons, muscle, and other tissue types. In keeping with this, a growing body of genetic, clinical, and pathological evidence indicates that RBP dysfunction and/or gene mutation leads to neurodegeneration and myopathy. Here, we summarize the current understanding of matrin 3 (MATR3), a poorly understood RBP implicated not only in ALS and frontotemporal dementia but also in distal myopathy. We begin by reviewing MATR3's functions, its regulation, and how it may be involved in both sporadic and familial neuromuscular disease. We also discuss insights gleaned from cellular and animal models of MATR3 pathogenesis, the links between MATR3 and other disease-associated RBPs, and the mechanisms underlying RBP-mediated disorders.

Lysine acetylation of NKG2D ligand Rae-1 stabilizes the protein and sensitizes tumor cells to NKG2D immune surveillance.

Shedding, loss of expression, or internalization of natural killer group 2, member D (NKG2D) ligands from the tumor cell surface leads to immune evasion, which is associated with poor prognosis in patients with cancer. In many cancers, matrix metalloproteinases cause the proteolytic shedding of NKG2D ligands. However, it remained unclear how to protect NKG2D ligands from shedding. Here, we showed that the shedding of the mouse NKG2D ligand Rae-1 can be prevented by two critical acetyltransferases, GCN5 and PCAF, which acetylate the lysine residues of Rae-1 to avoid shedding both in vitro and in vivo. In contrast, mutations at lysines 80 and 87 of Rae-1 abrogated this acetylation and thereby desensitized tumor cells to NKG2D-dependent immune surveillance. Notably, the protein levels of GCN5 correlated with the expression levels of the human NKG2D ligand ULPB1 in a human tumor tissue microarray and, more importantly, with prolonged overall survival in many cancers. Our results suggest that the acetylation of Rae-1 protein at lysines 80 and 87 by GCN5 and PCAF protects Rae-1 from shedding so as to activate NKG2D-dependent immune surveillance. This discovery may shed light on new targets for NKG2D immunotherapy in cancer treatment.

Molecular mechanism underlying selective inhibition of mRNA nuclear export by herpesvirus protein ORF10.

Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1-Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1-Nup98, the ORF10-Rae1-Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10-RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1-Nup98 in regulating host mRNA export.

SARS-CoV-2 Orf6 hijacks Nup98 to block STAT nuclear import and antagonize interferon signaling.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

Transport rate of EAAT2 is regulated by amino acid located at the interface between the scaffolding and substrate transport domains.

Excitatory Amino Acid Transporters (EAATs) are plasma membrane proteins responsible for maintenance of low extracellular concentrations of glutamate in the CNS. Dysfunction in their activity is implicated in various neurological disorders. Glutamate transport by EAATs occurs through the movement of the central transport domain relative to the scaffold domain in the EAAT membrane protein. Previous studies suggested that residues located within the interface of these two domains in EAAT2, the main subtype of glutamate transporter in the brain, are involved in regulating transport rates. We used mutagenesis, structure-function relationship, surface protein expression and electrophysiology studies, in transfected COS-7 cells and oocytes, to examine residue glycine at position 298, which is located within this interface. Mutation G298A results in increased transport rate without changes in surface expression, suggesting a more hydrophobic and larger alanine results in facilitated transport movement. The increased transport rate does not involve changes in sodium affinity. Electrophysiological currents show that G298A increase both transport and anion currents, suggesting faster transitions through the transport cycle. This work identifies a region critically involved in setting the glutamate transport rate.

Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain.

Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.

In situ 3D visualization of biomineralization matrix proteins.

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.

The nucleoskeleton protein IFFO1 immobilizes broken DNA and suppresses chromosome translocation during tumorigenesis.

Chromosome translocation is a major cause of the onset and progression of diverse types of cancers. However, the mechanisms underlying this process remain poorly understood. Here, we identified a non-homologous end-joining protein, IFFO1, which structurally forms a heterotetramer with XRCC4. IFFO1 is recruited to the sites of DNA damage by XRCC4 and promotes the repair of DNA double-strand breaks in a parallel pathway with XLF. Interestingly, IFFO1 interacts with lamin A/C, forming an interior nucleoskeleton. Inactivating IFFO1 or its interaction with XRCC4 or lamin A/C leads to increases in both the mobility of broken ends and the frequency of chromosome translocation. Importantly, the destruction of this nucleoskeleton accounts for the elevated frequency of chromosome translocation in many types of cancer cells. Our results reveal that the lamin A/C-IFFO1-constituted nucleoskeleton prevents chromosome translocation by immobilizing broken DNA ends during tumorigenesis.

Hexameric NuMA:LGN structures promote multivalent interactions required for planar epithelial divisions.

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.

Grafting a short chameleon sequence from αB crystallin into a ß-sheet scaffold protein.

Many protein and peptide sequences are self-assembled into ß-sheet-rich fibrous structures called amyloids. Their atomic details provide insights into fundamental knowledge related to amyloid diseases. To study the detailed structure of the amyloid, we have developed a model system that mimics the self-assembling process of the amyloid within a water-soluble protein, termed peptide self-assembly mimic (PSAM). PSAM enables capturing of a peptide sequence within a water-soluble protein, thus making structural and energetics-related studies possible. In this work, we extend our PSAM approach to a naturally occurring chameleon sequence from αB crystallin. We chose "Val-Leu-Gly-Asp-Val (VLGDV)", a five amino-acid sequence, which forms a ß-turn in the native structure and a ß-barrel in the amyloid oligomer cylindrin, as a grafting sequence to the PSAM scaffold. The crystal structure revealed that the sequence grafting induced ß-sheet bending at the grafted site. We further investigated the role of the central glycine residue and found that its role in the ß-sheet bending is dependent on the neighboring residues. The ability of PSAM to observe the structural alterations induced by the grafted sequence provides an opportunity to evaluate the structural impact of a sequence from the peptide self-assembly.

RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling.

RNF8 plays a critical role in DNA damage response (DDR) to initiate ubiquitination-dependent signaling. To better characterize the role of RNF8 in UV-induced DDR, we searched for novel substrates of RNF8 and identified NONO as one intriguing substrate. We found that: (i) RNF8 ubiquitinates NONO and (ii) UV radiation triggers NONO ubiquitination and its subsequent degradation. Depletion of RNF8 inhibited UV-induced degradation of NONO, suggesting that RNF8 targets NONO for degradation in response to UV damage. In addition, we found that 3 NONO lysine residues (positions 279, 290 and 295) are important for conferring its instability in UV-DDR. Depletion of RNF8 or expression of NONO with lysine to arginine substitutions at positions 279, 290 and 295 prolonged CHK1 phosphorylation over an extended period of time. Furthermore, expression of the stable mutant, but not wild-type NONO, induced a prolonged S phase following UV exposure. Stable cell lines expressing the stable NONO mutant showed increased UV sensitivity in a clonogenic survival assay. Since RNF8 recruitment to the UV-damaged sites is dependent on ATR, we propose that RNF8-mediated NONO degradation and subsequent inhibition of NONO-dependent chromatin loading of TOPBP1, a key activator of ATR, function as a negative feedback loop critical for turning off ATR-CHK1 checkpoint signaling in UV-DDR.

Heteromeric RNP Assembly at LINEs Controls Lineage-Specific RNA Processing.

Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.

Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization.

Abnormalities in nucleic acid processing are associated with the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in Matrin 3 (MATR3), a poorly understood DNA- and RNA-binding protein, cause familial ALS/FTD, and MATR3 pathology is a feature of sporadic disease, suggesting that MATR3 dysfunction is integrally linked to ALS pathogenesis. Using a rat primary neuron model to assess MATR3-mediated toxicity, we noted that neurons were bidirectionally vulnerable to MATR3 levels, with pathogenic MATR3 mutants displaying enhanced toxicity. MATR3's zinc finger domains partially modulated toxicity, but elimination of its RNA recognition motifs had no effect on survival, instead facilitating its self-assembly into liquid-like droplets. In contrast to other RNA-binding proteins associated with ALS, cytoplasmic MATR3 redistribution mitigated neurodegeneration, suggesting that nuclear MATR3 mediates toxicity. Our findings offer a foundation for understanding MATR3-related neurodegeneration and how nucleic acid binding functions, localization, and pathogenic mutations drive sporadic and familial disease.

Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble.

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

Protein⁻Protein Interactions with Connexin 43: Regulation and Function.

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.

Regulation of mitotic spindle assembly factor NuMA by Importin-ß.

Ran-guanosine triphosphatase orchestrates mitotic spindle assembly by modulation of the interaction between Importin-α/-ß and spindle assembly factors (SAFs). The inhibition of SAFs performed by importins needs to be done without much sequestration from abundant nuclear localization signal (NLS) -containing proteins. However, the molecular mechanisms that determine NLS-binding selectivity and that inhibit activity of Importin-ß-regulated SAFs (e.g., nuclear mitotic apparatus protein [NuMA]) remain undefined. Here, we present a crystal structure of the Importin-α-NuMA C terminus complex showing a novel binding pattern that accounts for selective NLS recognition. We demonstrate that, in the presence of Importin-α, Importin-ß inhibits the microtubule-binding function of NuMA. Further, we have identified a high-affinity microtubule-binding region that lies carboxyl-terminal to the NLS, which is sterically masked by Importin-ß on being bound by Importin-α. Our study provides mechanistic evidence of how Importin-α/-ß regulates the NuMA functioning required for assembly of higher-order microtubule structures, further illuminating how Ran-governed transport factors regulate diverse SAFs and accommodate various cell demands.

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function.

The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins.

Protein interactions involving intrinsically disordered proteins (IDPs) comprise a variety of binding modes, from the well-characterized folding upon binding to dynamic fuzzy complexes. To date, most studies concern the binding of an IDP to a structured protein, while the interaction between two IDPs is poorly understood. In this study, NMR, smFRET, and molecular dynamics (MD) simulation are combined to characterize the interaction between two IDPs, the C-terminal domain (CTD) of protein 4.1G and the nuclear mitotic apparatus (NuMA) protein. It is revealed that CTD and NuMA form a fuzzy complex with remaining structural disorder. Multiple binding sites on both proteins were identified by molecular dynamics and mutagenesis studies. This study provides an atomic scenario in which two IDPs bearing multiple binding sites interact with each other in dynamic equilibrium. The combined approach employed here could be widely applicable for investigating IDPs and their dynamic interactions.

Cell division orientation is coupled to cell-cell adhesion by the E-cadherin/LGN complex.

Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is mediated by the evolutionarily conserved LGN/NuMA complex, which regulates cortical attachments of astral spindle microtubules. We show that LGN, which adopts a three-dimensional structure similar to cadherin-bound catenins, binds directly to the E-cadherin cytosolic tail and thereby localizes at cell-cell adhesions. On mitotic entry, NuMA is released from the nucleus and competes LGN from E-cadherin to locally form the LGN/NuMA complex. This mediates the stabilization of cortical associations of astral microtubules at cell-cell adhesions to orient the mitotic spindle. Our results show how E-cadherin instructs the assembly of the LGN/NuMA complex at cell-cell contacts, and define a mechanism that couples cell division orientation to intercellular adhesion.