The EMBO Journal

Withdrawal StatementThe authors have withdrawn their manuscript because, upon reanalyzing the primary data it appears that the methods used by one of the authors, S.M., might have compromised the reliability of the data presented in Figure 1. We sincerely apologize for any harm or confusion this may cause to BioRxiv and its users.

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand GAS6 are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion (FA) turnover, cell spreading and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL but not TYRO3, and downstream activation of PI3K. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements and macropinocytosis that jointly contribute to cancer cell invasion.

VCP/p97 is an AAA+ ATPase that, together with its cofactors UFD1-NPL4 (p97-UN), binds and unfolds ubiquitylated substrates to maintain cellular homeostasis. The human p97-UN complex associates with additional cofactors, but how these cofactors modulate p97-UN activity is not fully understood. Here, we screen for cofactors that enhance p97-UN activity and identify FAF2 as the strongest activator. Using biochemical and structural approaches, we show how FAF2 engages p97-UN and polyubiquitin to promote unfolding. We define a conserved activation motif in FAF2 that contacts both UFD1 and the ubiquitin proximal to the initiator, stabilizing and initiating unfolding in a UFD1-dependent manner. We leverage the features of FAF2 AM to engineer de novo proteins that potently enhance unfolding, providing a rational strategy to boost p97 activity. Our findings reveal how cofactors can provide additional adaptive control, fine-tuning human p97 activity to unfold challenging substrates and those modified with short ubiquitin chains.

Selective autophagy restrains innate immune signalling to maintain tissue homeostasis, yet how this repression is rapidly relieved during infection remains unclear. Here, we show that under basal conditions the inhibitor of {kappa}B kinase {gamma} (IKK{gamma}) Kenny is sequestered at autophagosomes through Atg8 and the selective autophagy receptor Ref(2)P, thereby silencing Imd pathway activity. Bacterial infection disrupts this interaction, releasing the IKK complex to enable immune signalling. Mechanistically, we identify the initiator caspase Dredd as a direct interactor of the IKK{gamma} Kenny and show that Dredd binds and cleaves Kenny in a ubiquitination-dependent manner during infection. This cleavage removes an N-terminal LC3-interacting region, uncoupling the IKK complex from autophagosomal degradation. Dredd-mediated processing of Kenny stabilises the IKK complex and is required for activation of the NF-{kappa}B transcription factor Relish, robust antibacterial responses, and host survival following infection. Together, these findings uncover a mechanism by which caspase-mediated cleavage intersects with selective autophagy to dynamically control NF-{kappa}B signalling during bacterial infection. Short summaryBacterial infection activates NF-{kappa}B signalling by triggering caspase-dependent cleavage of the IKK subunit Kenny, releasing the IKK complex from autophagosomal repression to enable effective innate immune responses.

The repair of DNA double-strand breaks in repetitive sequences is challenging because the abundance of potential templates for homology-directed repair (HDR) exacerbates the risk of ectopic recombination and chromosome rearrangements. Relocalization of repair sites in repetitive sequences to a safe location prior to RAD51 loading has been observed in various organisms and is thought to suppress ectopic recombination and chromosomal rearrangements. We characterized this phenomenon in the rDNA repeats that reside within the nucleoli, specialized nuclear compartments where ribosome biosynthesis takes place. DSB induction in the rDNA repeats is associated with large-scale mobilization of the broken rDNA repeats from inside of the nucleoli to the nucleolar periphery where they are repaired by HDR. Here, we show that the two adaptor proteins Treacle and MDC1 are coordinating the sequence of events that ensue in response to rDNA break induction. Recruitment of the HDR machinery to rDNA breaks is dependent on the nucleolar adaptor Treacle, and specifically on its role in rDNA mobilization upon break induction. We demonstrate that following mobilization of the rDNA repeats and subsequent establishment of the {gamma}H2AX chromatin domain in the nucleolar periphery, the MDC1-RNF8-RNF168 axis is mediating the recruitment of the BRCA1-PALB2-BRCA2 complex and RAD51 loading. This coordinated sequence of events thus ensures that RAD51 loading is coupled to rDNA break mobilization to the nucleolar periphery, which may prevent ectopic recombination between repeats.

Proteins are typically targeted to the proteasome for degradation through the attachment of ubiquitin chains and the proteasome initiates degradation at a disordered region within the target protein. Yet some proteins with ubiquitin chains and disordered regions escape degradation. Here we investigate how the position of the ubiquitin chain on the target protein relative to the disordered region modulates degradation and show that the distance between the two determines whether a protein is degraded efficiently. This distance depends on the type of the degradation tag and is likely a result of the separation on the proteasome between the receptor that binds the tag and the site that engages the disordered region.

Primordial follicle oocyte activation (PFA) and zygotic genome activation (ZGA) represent two major waves of transcription activation respectively required for oocyte growth and preimplantation embryo development. Although many shared molecular hallmarks between PFA and ZGA suggest potential common factors and mechanisms driving both waves of transcriptional activation, such factors are yet to be identified. Here we demonstrate that the pioneer factor NFYA belongs to such regulators. Oocyte-specific Nfya deletion impairs open chromatin establishment and transcriptional activation during PFA, which triggers non-canonical ferroptosis leading to early folliculogenesis failure. Moreover, acute NFYA depletion in zygotes causes defective ZGA and predominantly two-cell embryo arrest. Mechanistically, although NFYA exhibits distinct chromatin-binding preferences predominantly targeting promoters during PFA and enhancers during ZGA, pre-occupied NFYA regulates chaperones and histone genes in both PFA and ZGA through conserved promoter binding. Together, our studies establish NFYA as a multifaceted regulator of genome activation during both PFA and ZGA. HighlightsO_LINFYA deficiency impairs primordial follicle oocyte activation (PFA) and triggers non-canonical ferroptosis resulting in early folliculogenesis failure C_LIO_LINFYA depletion impairs zygotic genome activation (ZGA) and causes predominantly 2-cell embryo arrest C_LIO_LIConserved and distinct NFYA-chromatin interactions drive both PFA and ZGA C_LIO_LIChaperones are pre-occupied and regulated by NFYA and their inhibition impairs both PFA and ZGA. C_LI

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a disproportionally greater impact on the overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites due to repetitive nature, clustering and high transcriptional activity. Notably, recent progress in understanding how the rDNA damage response is organized has highlighted the key role of adaptor proteins in the response. Here we identify that the scaffold and tumor suppressor, RASSF1A is recruited at sites of damage and particularly enriched at rDNA breaks. Employing targeted nucleolar DNA damage, we find that RASSF1A recruitment requires ATM activity and depends on the 53BP1. At sites of damage RASSF1A facilitates local ATM signal establishment and rDNA break repair. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Moreover, meta-analysis of a lung adenocarcinoma cohort showed that epigenetic silencing of the scaffold leads in rDNA copy number discrepancies. Overall, we present evidence that RASSF1A acts as a DNA repair factor and offer mechanistic insight in how the nucleolar DNA damage response is organized.

The proline-rich antimicrobial peptide (PrAMP) drosocin is produced by Drosophila species to combat bacterial infection. Unlike many PrAMPs, drosocin is O-glycosylated at threonine 11, a post-translation modification that enhances its antimicrobial activity. Here we demonstrate that the O-glycosylation influences not only cellular uptake of the peptide, but also interacts with its intracellular target, the ribosome. Cryo-electron microscopy structures of glycosylated drosocin on the ribosome at 2.1-2.8 [A] resolution reveal that the peptide interferes with translation termination by binding within the polypeptide exit tunnel and trapping RF1 on the ribosome, reminiscent of that reported for the PrAMP apidaecin. The glycosylation of drosocin enables multiple interactions with U2609 of the 23S rRNA, leading to conformational changes that break the canonical base-pair with A752. Collectively, our study provides novel molecular insights into the interaction of O-glycosylated drosocin with the ribosome, which provides a structural basis for future development of this class of antimicrobials.

To rapidly respond and adapt to stresses, such as viral infections, cells have evolved several mechanisms, which include the activation of stress response pathways and the innate immune response. These stress responses result in the rapid inhibition of translation and condensation of stalled mRNAs, together with RNA-binding proteins and signalling components, into cytoplasmic biocondensates called stress granules. Increasing evidence suggests that stress granules contribute to antiviral defense and thus viruses need to evade these response pathways to propagate. In addition, the stress granule pathway is proposed to be dynamic and adaptable to specific stresses. We previously showed that Feline Calicivirus (FCV) impairs SGs assembly by cleaving the scaffolding protein G3BP1. We also observed that uninfected bystander cells assembled G3BP1-granules, suggesting a paracrine response trigged by the infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of paracrine granules. They are different from canonical stress granules and exhibit specific kinetics of assembly-disassembly, protein and RNA composition and are linked to antiviral activity. We propose that this paracrine induction reflects a novel cellular defence mechanism to limit viral propagation and promote stress responses in bystander cells. Summary statementWe describe a novel type of paracrine induced RNA granules associated with viruses, highlighting how different stresses results in heterogeneous stress granule-like condensates with specific cellular functions.

The strategies adopted by viruses to reprogram the protein translation and quality control machineries to promote infection are poorly understood. Here, we discovered that the viral ubiquitin deconjugase (vDUB) encoded in the large tegument protein of Epstein- Barr virus (EBV) regulates ribosomal stress responses. The vDUB participates in protein complexes that include the ubiquitin ligases ZNF598 and LTN1 and the UFM1 ligase UFL1. Upon ribosomal stalling, the vDUB counteracts the ubiquitination of 40S ribosome subunits, inhibits the degradation of translation-stalled polypeptides by the proteasome, and prevents UFMylation of the 60S particle, which impairs the ER-phagy- dependent clearance of stalled products. Inhibition of the ribosome quality control activates a GCN2-dependent integrated stress response that decreases global protein translation while promoting the readthrough of stall-inducing mRNAs. The vDUB enhances viral mRNAs translation and virus release during productive infection, pointing to a pivotal role in cell reprogramming that enables virus production and underlies the pathogenesis of EBV-associated cancers and autoimmune diseases. GRAPHIC SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/526464v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c7421dorg.highwire.dtl.DTLVardef@c59b8dorg.highwire.dtl.DTLVardef@1676b4eorg.highwire.dtl.DTLVardef@b5e111_HPS_FORMAT_FIGEXP M_FIG C_FIG

During mitosis, properly aligned chromosomes stabilise microtubule ends with the help of kinetochores to ensure timely segregation of chromosomes. Microtubule-binding components of the human outer kinetochore, such as Ndc80 and Ska complexes, are present in multiple copies and together bind several microtubule ends, creating a highly multivalent binding interface. Whereas Ndc80:Ndc80 and Ndc80:microtubule binding is crucial for interface stability, Ndc80 alone in absence of Ska is unable to support stable kinetochore-attachments. Using cryoET, we demonstrate that oligomeric Ndc80:Ska assemblies stabilise microtubule ends against shortening by strengthening lateral contacts between tubulin protofilaments at microtubule plus-ends. We further identify a point mutation within the SKA1 microtubule-binding domain that does not affect microtubule-binding of individual Ska molecules, but does abolish Ska:Ska interactions. Finally, we report that oligomerisation of Ska, in a cooperative fashion together with the Ndc80, is necessary to maintain stable microtubule attachments both in vivo and in vitro.

Nuclear export of mRNAs requires loading the mRNP to the transporter Mex67/Mtr2 in the nucleoplasm, controlled access to the pore by the basket-localized TREX2 complex and mRNA release at the cytoplasmic site by the DEAD-box RNA helicase Dbp5. Asymmetric localisation of nucleoporins (NUPs) and transport components as well as the ATP dependency of Dbp5 ensure unidirectionality of transport. Trypanosomes possess homologues of the mRNA transporter Mex67/Mtr2, but not of TREX2 or Dbp5. Instead, nuclear export is likely fuelled by the GTP/GDP gradient created by the Ran GTPase. However, it remains unclear, how directionality is achieved since the current model of the trypanosomatid pore is mostly symmetric. We have revisited the architecture of the trypanosome nuclear pore complex using a novel combination of expansion microscopy, proximity labelling and streptavidin imaging. We could confidently assign the NUP76 complex, a known Mex67 interaction platform, to the cytoplasmic site of the pore. The resulting availability of reference proteins for basket, inner ring and cytoplasmic site allowed mapping of all 75 trypanosome proteins with known nuclear pore localisation to a sub-region of the pore based on mass spectrometry data from proximity labelling. This approach defined many further asymmetrically localised nuclear pore components. At the nuclear site, we identified several trypanosome-unique proteins, for instance the FG-NUPs NUP64/NUP98, but also proteins with structural homology to TREX-2 components. We mapped the components of the Ran-based nuclear export system and confirm the absence of a Dbp5 homologue. Lastly, we demonstrate, by deploying an auxin degron system, that NUP76 holds an essential role in mRNA export consistent with a functional orthology to NUP82/88. Altogether, the combination of proximity labelling with expansion microscopy revealed an asymmetric architecture of the trypanosome nuclear pore supporting inherent roles fort directed transport. Our approach delivered novel nuclear pore associated components inclusive positional information, which can now be interrogated for functional roles to explore trypanosome specific adaptions of the nuclear basket, export control and mRNP remodelling.

Targeted protein degradation through the CDC48 unfoldase enables the maintenance and rapid adaptation of proteomes across eukaryotes. However, the profound differences between animals, fungi, and plants are expected to have led to a significant adaptation of the CDC48-mediated degradation. While animal and fungal CDC48 systems have shown structural and functional preservation, such analysis is lacking for plants. We determined the structural and functional characteristics of Arabidopsis thaliana CDC48A in various states and bound to the target-identifying cofactors UFD1 and NPL4. Our analysis reveals several features that distinguish AtCDC48 from its animal and yeast counterparts, despite an 80% sequence identity. Key features are that AtCDC48A displays distinct domain dynamics and interacts differently with AtNPL4. Moreover, AtNPL4 and AtUFD1 do not form an obligate heterodimer, but independently bind to AtCDC48A and mediate target degradation; however, their joint action is synergistic. An evolutionary analysis supports that these Arabidopsis features are conserved across plants and represent the ancestral state of eukaryotic CDC48 systems. Jointly, our findings support that plant CDC48 retains a greater modular and combinatorial cofactor usage, highlighting a specific adaptation of targeted protein degradation in plants.

Surveillance of mRNA translation relies on a suite of ribosome-associated quality control pathways. Recently, a novel pathway induced by trapping of translation factors in the ribosomal A-site has been described, involving ubiquitination of RPS27A/eS31 by the human E3 ubiquitin ligase RNF25. Here, we show that not only ribosome-stalling by low doses of translation inhibitors, but also amino acid starvation induces RPS27A/eS31 ubiquitination, identifying a natural trigger of RNF25 activation. Even under optimal growth conditions, RNF25 senses and resolves transient ribosome stalls. RPS27A/eS31 ubiquitination specifically depends on the ribosome collision sensor GCN1, a known cofactor of GCN2 involved in the integrated stress response. RNF25 and GCN2 both possess a GCN1-binding RWD domain, indicating a competitive relationship, with GCN2 acting as a negative regulator of RNF25 activation. Although both RNF25 and GCN2 respond to amino acid starvation, RPS27A/eS31 ubiquitination by RNF25 is not required for GCN2 activation, showing that both act in independent pathways. We propose that the RNF25 pathway acts as a first line of defence to resolve ribosome collisions, outcompeted by GCN2 binding to GCN1 under acute stress.

In bacteria, chromosome segregation occurs progressively, from the origin to the terminus, a few minutes after the replication of each locus. In-between replication and segregation, sister loci are maintained in an apparent cohesive state by topological links. Whereas topoisomerase IV (Topo IV), the main bacteria decatenase, controls segregation, little is known regarding the influence of the cohesion step on chromosome folding. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, a massive chromosome reorganization takes place, associated with increases in trans-contacts between catenated sister chromatids and in long-range cis-contacts between the terminus and distant loci on the genome. A genetic analysis of these signals allowed us to decipher specific roles for Topo IV and Topo III, an accessory decatenase. Moreover we revealed the role of MatP, the terminus macrodomain organizing system and MukB, the E. coli SMC in organizing sister chromatids tied by persistent catenation links. We propose that large-scale conformation changes observed in these conditions reveal a defective decatenation hub located in the terminus area. Altogether, our findings support a model of spatial and temporal partition of the tasks required for sister chromosome segregation.

The capacity of host cells to sustain or restrict virus infection is influenced by their proteome. Understanding the compendium of proteins defining cellular permissiveness is key to many questions in fundamental virology. Here, we apply a multiomic approach to determine the proteins that are associated with highly permissive, intermediate, and hostile cellular states. We observed two groups of differentially regulated genes: i) with robust changes in mRNA and protein levels, and ii) with protein/RNA discordances. Many of the latter are classified as interferon stimulated genes (ISGs) but have no reported antiviral activity. This suggests that IFN-dependent changes in mRNA levels do not imply antiviral function. Phosphoproteomics revealed an additional regulatory layer involving non-signalling proteins with altered phosphorylation. Indeed, we confirmed that several permissiveness-associated proteins with changes in abundance or phosphorylation regulate infection fitness. Altogether, our study provides a comprehensive and systematic map of the cellular alterations driving virus susceptibility.

To ensure the correct euploid state of embryos, it is essential that vertebrate oocytes await fertilization arrested at metaphase of meiosis II. This MII arrest is mediated by XErp1/Emi2, which inhibits the ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome). Cyclin B3 in complex with Cdk1 (cyclin-dependent kinase 1) is essential to prevent an untimely arrest of vertebrate oocytes in meiosis I by targeting XErp1/Emi2 for degradation. Yet, the molecular mechanism of XErp1/Emi2 degradation in MI is not well understood. Here, by combining TRIM-Away in oocytes with egg extract and in vitro studies, we demonstrate that a hitherto unknown phosphate-binding pocket in cyclin B3 is essential for efficient XErp1/Emi2 degradation in meiosis I. This pocket enables Cdk1/cyclin B3 to bind pre-phosphorylated XErp1/Emi2 facilitating further phosphorylation events, which ultimately target XErp1/Emi2 for degradation in a Plk1 (Polo-like kinase 1) dependent manner. Key elements of this degradative mechanism are conserved in frog and mouse. Our studies identify a novel, evolutionarily conserved determinant of Cdk/cyclin substrate specificity essential to prevent an untimely oocyte arrest at meiosis I with catastrophic consequences upon fertilization.

Transposable elements (TEs) are mobile DNA sequences that make up a sizeable fraction of mammalian genomes yet are often tightly repressed by transcriptional and epigenetic mechanisms. During early development, epigenetic reprogramming selectively loosens TE repression, and TE transcription actively contributes to embryogenesis. This raises the question: how can embryos and embryonic stem cells (ESCs) tolerate TE expression without incurring widespread inflammation or DNA damage? Here, we reveal multiple mechanisms that prevent innate immune activation by TE-derived cytosolic DNA, including reduced cGAS/STING expression and signalling, dampening of Type I interferon responses by pluripotency factors, and post-transcriptional restriction of retrotransposition. These layers of protection are essential, as experimental perturbation triggers loss of ESC self-renewal and pluripotency. Our data explain how early development can be permissive to TE expression while safeguarding against harmful effects of TE activity.

Stress granules form in response to diverse cellular perturbations to sequester translation components until the stress is resolved. Stress granules are composed of RNA-protein assemblies in membrane delimited structures and must be rapidly disassembled to release components to allow translation to resume. Disassembly of stress granules formed in response to heat stress is dependent on ubiquitiylation of stress granule components such as G3BP1. Ubiquitylation of stress granule proteins recruits the AAA-ATPase p97 (also known as VCP) to enable ubiquitin-dependent disassembly of these structures. Loss of p97 activity leads to the persistence of stress granules and is implicated in several age-related neurodegenerative diseases. Here we show that p97 recruitment to stress granules is dependent on its ubiquitin binding co-factor p47. p47 translocates to stress granules in response to a variety of cellular stressors and is required for the recruitment of p97 to stress granules. Loss of p47 leads to an inhibition in stress granule disassembly. We further show that p47 associates with G3BP1 in response to heat stress in a ubiquitin-dependent manner. Taken together our data adds to the growing list of p97 adaptors that are implicated in the recruitment of p97 for dissolution of stress granules.