The EMBO Journal

Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active type I (/{beta}) and III ({lambda}) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signalling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knock-out prevented it. The JAK/ STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/ STAT signalling. In conclusion, our study 1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, 2) shows that Omicron infection protects cells from influenza A virus super-infection, and 3) indicates that BA.1 and BA.5 induce comparable antiviral states.

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Acute lysosome damage triggers the endolysosome damage response (ELDR) in order to co-ordinate vesicle repair or removal by autophagy (lysophagy). However, it is unclear whether persistent damage, as occurs after chronic challenge to lysosome integrity, triggers wider cellular responses. Here, we show that longitudinal treatment with a lysosomotropic cancer therapeutic, the CDK4/6 inhibitor Palbociclib, invokes chronic lysosome damage in breast and lung cancer cells. Autophagy ameliorates but does not avert this phenotype, which persists over days. Damaged lysosomes form contacts with mitochondria, which correlates with mitochondrial stress and cytosolic efflux of immunostimulatory mitochondrial nucleic acids. Importantly, mitochondrial nucleic acid release is necessary for the anti-cancer interferon response to Palbociclib. In conclusion, chronic lysosome damage rewires cellular signalling responses in a mitochondrion-dependent manner and this effect should be considered when assessing the cellular actions of cancer therapeutics. Summary statementBozic et al suggest that lysosome damage can trigger interferon responses dependent upon mitochondrial release of immunogenic nucleic acid. This is associated with damaged lysosome-mitochondrion contacts and is prevented by autophagy.

Innate defences of the respiratory epithelium are the first barrier against incoming respiratory viruses. To understand the contribution of both basal (tonic) and induced interferon (IFN) to antiviral defences in a physiologically relevant system, we established air-liquid interface (ALI) cultures of primary human bronchial epithelium (HBE) and small airway epithelium (HSE). Via an organoid intermediate stage, the limited healthy donor material was expanded while preserving stemness and subsequently differentiated. Characterisation by spatial and transcriptomic analyses showed that the cellular diversity and architecture of our ALI cultures were comparable to native human lung epithelium. Upon infection with relevant human respiratory pathogens, such as Human Rhinovirus (HRV16) and human Coronaviruses (229E and NL63), only HRV16 induced a strong and early type I and III IFN response, leading to its eventual clearance from the cultures. Depletion of tonic type I/III IFNs using neutralising antibodies or scavengers reduced expression of levels of IFN-stimulated genes and increased infectious HRV production by [~]7-10-fold. Taken together, we present a method for generating primary lung epithelial cultures that retain their IFN status, demonstrate clearance of HRV by innate defences, and highlight the importance of tonic IFN in early antiviral defences. IMPORTANCEMild respiratory viral infections, for example, with human common cold coronaviruses or rhinoviruses, are a massive cause of human morbidity. The respiratory tract is the primary entry route for these viruses and also the contact site for initial innate immune defences. Here, we show that primary human lung epithelial cell-derived air-liquid interface cultures mimic the architecture and cell composition of native human lung epithelium, and retain both induced and tonic interferon (IFN) responses. Notably, our data show that the models innate immune defences are sufficient to clear human Rhinovirus (HRV) infections, which are characterised by rapid and robust IFN responses. Finally, depletion of tonic IFNs led to a marked increase in HRV infection. Thus, our research suggests that tonic low levels of IFNs contribute to the epithelial defence against viruses, maintaining the tissues immune readiness. Failure to maintain these tonic IFN levels increases the susceptibility towards infections.

The pandemic spread of SARS-CoV-2, the etiological agent of COVID-19, represents a significant and ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41{degrees}C. Fever is an evolutionarily conserved host response to microbial infection and inflammation that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 tropism and replication. Utilizing a 3D air-liquid interface (ALI) model that closely mimics the natural tissue physiology and cellular tropism of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. We show that temperature elevation induces wide-spread transcriptome changes that impact upon the regulation of multiple pathways, including epigenetic regulation and lncRNA expression, without disruption of general cellular transcription or the induction of interferon (IFN)-mediated antiviral immune defences. Respiratory tissue incubated at temperatures >37{degrees}C remained permissive to SARS-CoV-2 infection but severely restricted the initiation of viral transcription, leading to significantly reduced levels of intraepithelial viral RNA accumulation and apical shedding of infectious virus. To our knowledge, we present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication. Our data identify an important role for temperature elevation in the epithelial restriction of SARS-CoV-2 that occurs independently of the induction of canonical IFN-mediated antiviral immune defences and interferon-stimulated gene (ISG) expression.

Perturbations in biomolecular condensates that form by phase-transitioning are linked to a growing number of degenerative diseases. For example, mutations in a multivalent interaction network of the Ankyrin (ANK) and sterile alpha motif (SAM) domain-containing ANKS3 and ANKS6 proteins with the RNA-binding protein Bicaudal-C1 (Bicc1) associate with laterality defects and chronic kidney diseases known as ciliopathies. However, insights into the mechanisms that control RNA condensation in ribonucleoprotein particles (RNPs) are scarce. Here, we asked whether heterooligomerization modulates Bicc1 binding to RNA. Reconstitution assays in vitro and live imaging in vivo show that a K homology (KH) repeat of Bicc1 self-interacts and synergizes with SAM domain self-polymerization independently of RNA to concentrate bound mRNAs in gel-like granules that can split or fuse with each other. Importantly, emulsification of Bicc1 by ANKS3 inhibited binding to target mRNAs, whereas condensation by ANKS6 co-recruitment increased it by liberating the KH domains from ANKS3. Our findings suggest that the perturbation of Bicc1-Anks3-Anks6 RNP dynamics is a likely cause of associated ciliopathies.

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) induce cytotoxicity in homologous recombination repair (HRR)-deficient cancers by causing PARP1 to become trapped on chromatin, resulting in irreparable replication-associated DNA damage. Although increased clearance of trapped PARP1 from chromatin reduces the sensitivity of cancer cells to PARPi, details surrounding this process remain unclear. PARPi exposure is known to cause increased autophagy flux, whilst autophagy inhibition can hypersensitise cells to PARPi. Using various biochemical, cell biological and live imaging-based assays, we found that trapped PARP1 is cleared by nucleophagy, the selective autophagy of nuclear substrates. Specifically, the nucleophagy of trapped PARP1 was orchestrated by the selective autophagy receptor TEX264 and its partner segregase p97/VCP. TEX264 mediates this process by directly interacting with trapped PARP1, thus bridging PARP1 to the autophagosomal resident protein LC3 for processing via autophagy. Impeding this process, either chemically or genetically, heightened PARP1 trapping, leading to accumulation of protein aggregates, replication-associated DNA damage and cell lethality, re-sensitising PARPi-resistant cells to various PARPi. In conclusion, we show that nucleophagy acts in a cytoprotective manner to directly target PARPi-induced trapped PARP1 for degradation.

Emerging studies hint at the roles of autophagy-related proteins in various cellular processes in a eukaryotic cell. To understand if autophagy-related proteins influence genome stability, we examined a cohort of 35 autophagy mutants in Saccharomyces cerevisiae. We observed cells lacking Atg11 show poor mitotic stability of minichromosomes. Atg11 molecules dynamically localize to the spindle pole bodies (SPBs). Loss of Atg11 leads to a delayed cell cycle progression. Such cells accumulate at metaphase at an elevated temperature that is relieved when the spindle assembly checkpoint is inactivated. Indeed, atg11{Delta} cells have stabilized securin levels, that prevent anaphase onset, confirming chromosome biorientation defects associated with the mutant. Atg11 functions in the Kar9-dependent spindle positioning pathway and maintains Kar9 asymmetry by facilitating proper dynamic instability of astral microtubules (aMTs). Taken together, this study uncovers a non-canonical role of Atg11 in facilitating MT dynamics crucial for chromosome segregation.

Ribosome biogenesis involves the processing of precursor ribosomal RNAs (pre-rRNAs) and sequential assembly with ribosomal proteins. Here we report that nutrient deprivation severely impairs pre-rRNA processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, the accumulated pre-rRNAs are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient deprivation leads to perturbed ribosome assembly during nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine was dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which was in turn dependent on Raf/MEK/ERK signalling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that cancer cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.

Cohesin-NIPBL complexes extrude genomic DNA into loops that are constrained by CTCF boundaries. This process has important regulatory functions and weakens the separation between euchromatic and heterochromatic compartments. Cohesin can also bind PDS5A or PDS5B, which do not support loop extrusion but are required for the formation of CTCF boundaries. How PDS5 proteins perform this function is unknown. Here we show by in vitro single-molecule imaging that PDS5 proteins stop loop extrusion by facilitating the dissociation of NIPBL from cohesin. Hi-C experiments suggest that this function is required for the establishment of CTCF boundaries in cells. In silico modelling indicates that PDS5 proteins enable the separation between compartments by limiting cohesins velocity and chromatin-residence time. The degree of this compartmentalization depends on the frequency with which chromatin is extruded relative to the time it takes for compartments to form. These results identify PDS5 proteins as key regulators of genome organization. HighlightsO_LIPDS5 proteins stop loop extrusion by facilitating dissociation of NIPBL from cohesin. C_LIO_LIPDS5 proteins strengthen CTCF boundaries by limiting the lifetime of cohesin-NIPBL. C_LIO_LIPDS5 proteins regulate compartmentalization. C_LIO_LICompartmentalization is governed by polymer relaxation and loop extrusion dynamics. C_LI

The coil-coil rod domain mediating the lateral assembly of lamin filaments has been reported to undergo phosphorylation by proteomic approaches, but the functional implications of these modifications are currently unknown. Here, we report that serine 210 (S210) within the Lamin B1 rod domain is a mitotic phospho-acceptor residue controlled by the combined activities of the autophagy-activating kinase ULK1 and the mitotic kinases Aurora A and PLK1. The detection of Lamin B1 phospho-S210 with a specific phospho-antibody revealed its enrichment at the mitotic spindle and its association with a network of proteins with known functions in spindle assembly. Abrogation of the phosphorylation of the S210A Lamin B1 variant leads to an increase in the number of cells with multipolar or shorter spindles. We propose that mitotic phosphorylation of Lamin B1 S210 within the rod domain contributes to the maintenance of proper spindle function.

Gut microbiota exert an evolutionarily conserved influence on ageing, from invertebrates to humans. How do microbes that are physically confined to the gut lumen affect the systemic physiological process of ageing? In female Drosophila, we show that microbiota increase expression of the peptide hormone Tachykinin (Tk), which corresponds to reduced lifespan. Tk is required for microbiota to shorten lifespan, with knockdown rendering flies constitutively long-lived even in the presence of an intact microbiota. This lifespan extension does not come with canonical costs to fecundity or feeding, but impacts on triacylglyceride (TAG) storage suggest adaptive functions in metabolic homeostasis. In flies with defined (gnotobiotic) microbiotas, we show that we can model Tk-dependent effects of microbiota on lifespan and TAG by monoassociation with Acetobacter pomorum. These effects require Tk in the midgut, and the cognate TK receptor TkR99D in neurons, implicating a microbiota-gut-neuron relay. This relay also appears to compromise gut barrier function in aged flies, indicating roles in healthspan as well as lifespan. However, the effect of TkR99D is independent of its reported role in insulin signalling and adipokinetic hormone signalling which, respectively, are canonical regulators of lifespan and TAG metabolism, suggesting a non-canonical role for TkR99D elsewhere in the nervous system. Altogether our results implicate a microbiota-gut-neuron axis in ageing, via a specific bacterium modulating activity of a specific and evolutionarily-conserved hormone.

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.

Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria together with mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here we show that mtDNA depletion after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, is due to an exacerbated mtDNA turnover. Targeting of nucleoids is controlled by Twinkle which, together with the mitochondrial transmembrane proteins ATAD3 and SAMM50, orchestrate mitochondrial membrane remodeling to form extrusions. mtDNA removal depends on autophagy and requires the vesicular trafficking protein VPS35 which binds to Twinkle-enriched mitochondrial subcompartments upon mtDNA damage. Stimulation of autophagy by rapamycin selectively removes mtDNA deletions which accumulated during muscle regeneration in vivo, but without affecting mtDNA copy number. With these results we unveil a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network. We reveal the molecular targets involved in a process with multiple potential benefits against human mtDNA related diseases, either inherited, acquired or due to normal ageing.

Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors associate with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5-3 exoribonuclease XRN1 interact with enhancer of mRNA decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein (RNP) granules that are often enriched with mRNAs targeted for decay, such as microRNA (miRNA)-targeted mRNAs, yet paradoxically are not required for mRNA decay to occur. In this study, we show that disrupting the interaction between XRN1 and EDC4 or altering their stoichiometry leads to an inhibition of mRNA decapping, with miRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are directly responsible for preventing mRNA decapping under these conditions. Finally, we demonstrate that P-bodies act to support cell viability and prevent stress granule formation under conditions when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5-3 decay in human cells. HIGHLIGHTSO_LIXRN1-EDC4 interaction couples mRNA decapping with mRNA decay. C_LIO_LIDisrupting XRN1-EDC4 contact generates larger P-bodies that, in turn, inhibit decapping. C_LIO_LIP-bodies support cellular fitness in the absence of XRN1. C_LI

SMC complexes have ubiquitous roles in chromosome organisation. In Escherichia coli, the interplay between the SMC complex, MukBEF, and matS-bound MatP in the replication termination region, ter, results in depletion of MukBEF from ter, thus promoting chromosome individualisation by directing replichores to separate cell halves. MukBEF also interacts with topoisomerase IV ParC2E2 heterotetramers, to direct its chromosomal distribution to mirror that of MukBEF, thereby facilitating coordination between chromosome organisation and decatenation by topoisomerase IV. Here we demonstrate that the MukB dimerization hinge binds ParC and MatP with the same dimer to dimer stoichiometry. MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding. Furthermore, the MukB hinge fails to stably associate with matS-bound MatP, while MatP mutants deficient in matS binding are impaired in MukB hinge binding, demonstrating that mats competes with the hinge for MatP binding. Cells expressing MukBEF complexes containing a mutation in the MukB hinge interface for ParC/MatP binding are deficient in ParC binding in vivo, despite having a Muk+ topoisomerase IV+ phenotype. This mutant protein is also impaired in MatP binding in vitro, and cells expressing this variant exhibit a MukBEF cellular localisation consistent with impaired MatP binding.

Respiratory viruses such as influenza A virus (IAV) and SARS-CoV-2 (Covid-19) cause pandemic infections where cytokine storm syndrome, lung inflammation and pneumonia lead to high mortality. Given the high social and economic cost of these viruses, there is an urgent need for a comprehensive understanding of how the airways defend against virus infection. Viruses entering cells by endocytosis are killed when delivered to lysosomes for degradation. Lysosome delivery is facilitated by non-canonical autophagy pathways that conjugate LC3 to endo-lysosome compartments to enhance lysosome fusion. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that non-canonical autophagy protects mice from lethal IAV infection of the airways. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity murine-adapted IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV infection was controlled within epithelial barriers where non-canonical autophagy slowed fusion of IAV with endosomes and reduced activation of interferon signalling. This was consistent with conditional mouse models and ex vivo analysis showing that protection against IAV infection of lung was independent of phagocytes and other leukocytes. This establishes non-canonical autophagy pathways in airway epithelial cells as a novel innate defence mechanism that can restrict IAV infection and lethal inflammation at respiratory surfaces.

Nucleolar stress (NS) has been associated to several age-related diseases such as cancer or neurodegeneration. To investigate the mechanisms of toxicity triggered by NS, we here used (PR)n arginine-rich peptides that are found in patients of some neurodegenerative diseases. Although these peptides accumulate at nucleoli and generate NS, how this translates into cellular toxicity is poorly understood. We here reveal that whereas (PR)n expression leads to an overall decrease in protein abundance, this occurs concomitant with an accumulation of free ribosomal (r) proteins in the cytoplasm, a hallmark of ribosomopathies. Conversely, cells with acquired resistance to (PR)n peptides present global downregulation of r-proteins and low levels of mTOR signaling. In mice, systemic expression of (PR)97 drives widespread NS and accelerated ageing, which is associated to an increased expression of r-proteins and mTOR hyperactivation. Furthermore, the reduced lifespan of (PR)97-expressing mice was alleviated by the mTOR inhibitor rapamycin. Importantly, we show that the generalised accumulation of free r-proteins is a common outcome in response to chemical or genetic perturbations that trigger NS, such as Actinomycin D, TIF-IA depletion, or the expression of mutant HMGB1 variants recently associated to rare human diseases. Together, our study presents in vivo evidence supporting the role of NS as a driver of ageing, and provides a general framework to explain the toxicity caused by NS in mammalian cells.

The mammalian genome, organised into chromatin, adopts a three-dimensional (3D) folding within the cell nucleus with spatially segregated active and repressed compartments, termed A and B. However, how nucleosome deposition impacts these levels of organisation is unknown. Here, we monitored changes in 3D genome folding by Hi-C after impairing the chaperone HIRA, involved in histone H3.3 deposition. In the absence of HIRA, H3.3 enrichment decreases in compartment A that also shows weaker interactions. At this scale, histone post-translational modifications (PTMs) do not follow H3.3 changes. In line with impaired H3.3 nucleosome maintenance, compartment A accessibility measured by ATAC-seq increases. Specifically, at active genes, accessibility increases in gene bodies but decreases at promoters where compensation by H3.1 reduces nucleosome turnover. Notably, regions flanking active genes show reduced insulation. We conclude that the HIRA-dependent pathway involved in H3.3 deposition is key to maintain higher order organisation in active regions and impact compartmentalisation independently of histone PTMs.

Ovarian aging is closely associated with a decline in fertility and an increase in reproductive dysfunction. Ovarian granulosa cells (GCs) support oocyte homeostasis and development, yet insight into GC dysfunction during aging is limited. Here, we show that aged GCs of humans and mice have indications of elevated ferroptosis, including increased ferroptosis-related metabolites, lipid peroxidation, and iron accumulation. The ferroptosis inhibitor Ferrostatin-1 reversed ovarian impairment and fertility of aged mice in vivo. We show that the age-related reduction in the expression of TXN (thioredoxin) leads to ferroptosis in human and mouse GCs by blocking BNIP3L-dependent mitophagy. Exogenous activation of TXN could promote mitophagy, thereby clearing excessive ROS and inhibiting ferroptosis. These results suggest that anti-ferroptosis-related treatments may assist in treating aging-related reproductive disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/664497v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1b97e8forg.highwire.dtl.DTLVardef@123f8d0org.highwire.dtl.DTLVardef@a44c61org.highwire.dtl.DTLVardef@92b32e_HPS_FORMAT_FIGEXP M_FIG C_FIG Key pointsO_LIFerroptosis signatures are upregulated in aged GCs from human and mouse ovaries C_LIO_LITXN is deregulated in aged GCs, leading to mitochondrial and ROS metabolic dysfunction C_LIO_LITXN binds to DNA to regulate autophagy and mitophagy genes, including BNIP3L C_LIO_LIInhibition of ferroptosis can ameliorate GC dysfunction C_LI