Mitochondria and pathogen immunity: from killer to firestarter
Serving as an innate defence mechanism, invading pathogens elicit a broad inflammatory response in cells. In this issue, Brokatzky et al (2019) report that pathogens can cause activation of BAX/BAK which permeabilises a limited number of mitochondria. Induction of DNA damage, or release of mtDNA, triggers STING-dependent pro-inflammatory cytokine expression and secretion, revealing an unexpected role for the mitochondrial apoptotic machinery in immune defence. (Source: EMBO Journal)
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Riley, J. S., Tait, S. W. Tags: Autophagy & Cell Death, Microbiology, Virology & Host Pathogen Interaction News [amp ] Views Source Type: research

Phospho-RNA-seq: a modified small RNA-seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma
We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA-seq protocols due to lack of 5' phosphate or presence of 3' phosphate. These fragments were revealed using a modified protocol ("phospho-RNA-seq") incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5' and 3' ends, as well as human plasma exRNA. Analyzing phospho-RNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Giraldez, M. D., Spengler, R. M., Etheridge, A., Goicochea, A. J., Tuck, M., Choi, S. W., Galas, D. J., Tewari, M. Tags: RNA Biology Resource Source Type: research

A non-death function of the mitochondrial apoptosis apparatus in immunity
Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines,...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Brokatzky, D., Dörflinger, B., Haimovici, A., Weber, A., Kirschnek, S., Vier, J., Metz, A., Henschel, J., Steinfeldt, T., Gentle, I. E., Häcker, G. Tags: Autophagy & Cell Death, Immunology Articles Source Type: research

Human keratin 1/10-1B tetramer structures reveal a knob-pocket mechanism in intermediate filament assembly
To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob ...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Eldirany, S. A., Ho, M., Hinbest, A. J., Lomakin, I. B., Bunick, C. G. Tags: Cell Adhesion, Polarity & Cytoskeleton, Structural Biology Articles Source Type: research

Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance
Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STUbLs select specific substrates among myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term "ubiquitin hotspots". Ubiquitylation at th...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Höpfler, M., Kern, M. J., Straub, T., Prytuliak, R., Habermann, B. H., Pfander, B., Jentsch, S. Tags: Chromatin, Epigenetics, Genomics & Functional Genomics, Post-translational Modifications, Proteolysis & Proteomics Articles Source Type: research

Triggering MSR1 promotes JNK-mediated inflammation in IL-4-activated macrophages
Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Guo, M., Härtlova, A., Gierlinski, M., Prescott, A., Castellvi, J., Losa, J. H., Petersen, S. K., Wenzel, U. A., Dill, B. D., Emmerich, C. H., Ramon Y Cajal, S., Russell, D. G., Trost, M. Tags: Immunology, Post-translational Modifications, Proteolysis & Proteomics, Signal Transduction Articles Source Type: research

Pten controls B-cell responsiveness and germinal center reaction by regulating the expression of IgD BCR
In contrast to other B-cell antigen receptor (BCR) classes, the function of IgD BCR on mature B cells remains largely elusive as mature B cells co-express IgM, which is sufficient for development, survival, and activation of B cells. Here, we show that IgD expression is regulated by the forkhead box transcription factor FoxO1, thereby shifting the responsiveness of mature B cells towards recognition of multivalent antigen. FoxO1 is repressed by phosphoinositide 3-kinase (PI3K) signaling and requires the lipid phosphatase Pten for its activation. Consequently, Pten-deficient B cells expressing knock-ins for BCR heavy and li...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Setz, C. S., Khadour, A., Renna, V., Iype, J., Gentner, E., He, X., Datta, M., Young, M., Nitschke, L., Wienands, J., Maity, P. C., Reth, M., Jumaa, H. Tags: Immunology, Signal Transduction Articles Source Type: research

Cancer cells induce immune escape via glycocalyx changes controlled by the telomeric protein TRF2
In this study, we describe a mechanism by which cancer cells control MDSCs in human cancers by upregulating TRF2, a protein required for telomere stability. Specifically, we showed that the TRF2 upregulation in cancer cells has extratelomeric roles in activating the expression of a network of genes involved in the biosynthesis of heparan sulfate proteoglycan, leading to profound changes in glycocalyx length and stiffness, as revealed by atomic force microscopy. This TRF2-dependent regulation facilitated the recruitment of MDSCs, their activation via the TLR2/MyD88/IL-6/STAT3 pathway leading to the inhibition of natural kil...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Cherfils-Vicini, J., Iltis, C., Cervera, L., Pisano, S., Croce, O., Sadouni, N., Gyorffy, B., Collet, R., Renault, V. M., Rey-Millet, M., Leonetti, C., Zizza, P., Allain, F., Ghiringhelli, F., Soubeiran, N., Shkreli, M., Vivier, E., Biroccio, A., Gilson, Tags: Cancer, Cell Adhesion, Polarity & Cytoskeleton, Immunology Articles Source Type: research

Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis
Cells going through mitosis undergo precisely timed changes in cell shape and organisation, which serve to ensure the fair partitioning of cellular components into the two daughter cells. These structural changes are driven by changes in actin filament and microtubule dynamics and organisation. While most evidence suggests that the two cytoskeletal systems are remodelled in parallel during mitosis, recent work in interphase cells has implicated the centrosome in both microtubule and actin nucleation, suggesting the potential for regulatory crosstalk between the two systems. Here, by using both in vitro and in viv...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Farina, F., Ramkumar, N., Brown, L., Samandar Eweis, D., Anstatt, J., Waring, T., Bithell, J., Scita, G., Thery, M., Blanchoin, L., Zech, T., Baum, B. Tags: Cell Adhesion, Polarity & Cytoskeleton, Cell Cycle Articles Source Type: research

Actin filaments regulate microtubule growth at the centrosome
The centrosome is the main microtubule-organizing centre. It also organizes a local network of actin filaments. However, the precise function of the actin network at the centrosome is not well understood. Here, we show that increasing densities of actin filaments at the centrosome of lymphocytes are correlated with reduced amounts of microtubules. Furthermore, lymphocyte activation resulted in disassembly of centrosomal actin and an increase in microtubule number. To further investigate the direct crosstalk between actin and microtubules at the centrosome, we performed in vitro reconstitution assays based on (i) purif...
Source: EMBO Journal - June 3, 2019 Category: Molecular Biology Authors: Inoue, D., Obino, D., Pineau, J., Farina, F., Gaillard, J., Guerin, C., Blanchoin, L., Lennon-Dumenil, A.-M., Thery, M. Tags: Cell Adhesion, Polarity & Cytoskeleton, Immunology Articles Source Type: research

Chopping GSDMD: caspase-8 has joined the team of pyroptosis-mediating caspases
nflammatory and apoptotic caspases mediate two distinct forms of cell death: pyroptosis and apoptosis, respectively. Three independent studies have now demonstrated that the "apoptotic" caspase-8 can cleave gasdermin D (GSDMD) leading to pyroptosis-like cell death and IL-1β release in murine macrophages (Orning et al, 2018; Sarhan et al, 2018; Chen et al, 2019). Orning et al and Chen/Demarco et al also show that the NLRP3 inflammasome is activated downstream of active caspase-8, but they attribute this inflammasome activation to different pore-forming proteins, GSDMD and pannexin-1, ...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Gram, A. M., Booty, L. M., Bryant, C. E. Tags: Autophagy & Cell Death, Immunology News [amp ] Views Source Type: research

Helping daughters succeed: asymmetric distribution of glucose transporter mRNA
Rapidly proliferating cells growing by glucose fermentation must first transport glucose into the cell. Both budding yeast and human tumor cells utilize members of a conserved family of glucose transporters. In this issue of The EMBO Journal, Stahl et al (2019) reveal that budding yeast cells confer a growth advantage to their daughters using a novel mechanism, the asymmetric distribution to the daughter cell of the mRNA for a specific glucose transporter. (Source: EMBO Journal)
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: O'Donnell, A. F., Schmidt, M. C. Tags: Cell Cycle, Membrane & Intracellular Transport, Metabolism News [amp ] Views Source Type: research

Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4
(Source: EMBO Journal)
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Scherz-Shouval, R., Shvets, E., Fass, E., Shorer, H., Gil, L., Elazar, Z. Tags: Corrigendum Source Type: research

Extrinsic and intrinsic apoptosis activate pannexin-1 to drive NLRP3 inflammasome assembly
Pyroptosis is a form of lytic inflammatory cell death driven by inflammatory caspase-1, caspase-4, caspase-5 and caspase-11. These caspases cleave and activate the pore-forming protein gasdermin D (GSDMD) to induce membrane damage. By contrast, apoptosis is driven by apoptotic caspase-8 or caspase-9 and has traditionally been classified as an immunologically silent form of cell death. Emerging evidence suggests that therapeutics designed for cancer chemotherapy or inflammatory disorders such as SMAC mimetics, TAK1 inhibitors and BH3 mimetics promote caspase-8 or caspase-9-dependent inflammatory cell death and NLRP3 inflamm...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Chen, K. W., Demarco, B., Heilig, R., Shkarina, K., Boettcher, A., Farady, C. J., Pelczar, P., Broz, P. Tags: Autophagy & Cell Death, Immunology Articles Source Type: research

Single-cell damage elicits regional, nematode-restricting ethylene responses in roots
Plants are exposed to cellular damage by mechanical stresses, herbivore feeding, or invading microbes. Primary wound responses are communicated to neighboring and distal tissues by mobile signals. In leaves, crushing of large cell populations activates a long-distance signal, causing jasmonate production in distal organs. This is mediated by a cation channel-mediated depolarization wave and is associated with cytosolic Ca2+ transient currents. Here, we report that much more restricted, single-cell wounding in roots by laser ablation elicits non-systemic, regional surface potential changes, calcium waves, and reactive oxyge...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Marhavy, P., Kurenda, A., Siddique, S., Denervaud Tendon, V., Zhou, F., Holbein, J., Hasan, M. S., Grundler, F. M., Farmer, E. E., Geldner, N. Tags: Plant Biology, Signal Transduction Articles Source Type: research

OTUB1 inhibits CNS autoimmunity by preventing IFN-{gamma}-induced hyperactivation of astrocytes
Astrocytes are critical regulators of neuroinflammation in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Growing evidence indicates that ubiquitination of signaling molecules is an important cell-intrinsic mechanism governing astrocyte function during MS and EAE. Here, we identified an upregulation of the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) in astrocytes during MS and EAE. Mice with astrocyte-specific OTUB1 ablation developed more severe EAE due to increased leukocyte accumulation, proinflammatory gene transcription, and demyelination in the spinal...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Wang, X., Mulas, F., Yi, W., Brunn, A., Nishanth, G., Just, S., Waisman, A., Brück, W., Deckert, M., Schlüter, D. Tags: Immunology, Neuroscience Articles Source Type: research

In situ and high-resolution cryo-EM structure of a bacterial type VI secretion system membrane complex
Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. It is composed of a phage tail-like structure inserted in the bacterial cell envelope by a membrane complex (MC) comprising the TssJ, TssL and TssM proteins. We previously reported the low-resolution negative-stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tom...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Rapisarda, C., Cherrak, Y., Kooger, R., Schmidt, V., Pellarin, R., Logger, L., Cascales, E., Pilhofer, M., Durand, E., Fronzes, R. Tags: Microbiology, Virology & Host Pathogen Interaction, Structural Biology Articles Source Type: research

The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state
The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the func...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Tavoulari, S., Thangaratnarajah, C., Mavridou, V., Harbour, M. E., Martinou, J.-C., Kunji, E. R. Tags: Membrane & Intracellular Transport Articles Source Type: research

Asymmetric distribution of glucose transporter mRNA provides a growth advantage in yeast
Asymmetric localization of mRNA is important for cell fate decisions in eukaryotes and provides the means for localized protein synthesis in a variety of cell types. Here, we show that hexose transporter mRNAs are retained in the mother cell of S. cerevisiae until metaphase–anaphase transition (MAT) and then are released into the bud. The retained mRNA was translationally less active but bound to ribosomes before MAT. Importantly, when cells were shifted from starvation to glucose-rich conditions, HXT2 mRNA, but none of the other HXT mRNAs, was enriched in the bud after MAT. This enrichment was dependent on the ...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Stahl, T., Hümmer, S., Ehrenfeuchter, N., Mittal, N., Fucile, G., Spang, A. Tags: Cell Cycle, Membrane & Intracellular Transport, Metabolism Articles Source Type: research

YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of satellite cells
Skeletal muscle satellite cells (SCs) are adult muscle stem cells responsible for muscle regeneration after acute or chronic injuries. The lineage progression of quiescent SC toward activation, proliferation, and differentiation during the regeneration is orchestrated by cascades of transcription factors (TFs). Here, we elucidate the function of TF Yin Yang1 (YY1) in muscle regeneration. Muscle-specific deletion of YY1 in embryonic muscle progenitors leads to severe deformity of diaphragm muscle formation, thus neonatal death. Inducible deletion of YY1 in SC almost completely blocks the acute damage-induced muscle repair a...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Chen, F., Zhou, J., Li, Y., Zhao, Y., Yuan, J., Cao, Y., Wang, L., Zhang, Z., Zhang, B., Wang, C. C., Cheung, T. H., Wu, Z., Wong, C. C.-L., Sun, H., Wang, H. Tags: Metabolism, Stem Cells, Transcription Articles Source Type: research

Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza
The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degra...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Park, E.-S., Byun, Y. H., Park, S., Jang, Y. H., Han, W.-R., Won, J., Cho, K. C., Kim, D. H., Lee, A. R., Shin, G.-C., Park, Y. K., Kang, H. S., Sim, H., Ha, Y. N., Jae, B., Son, A., Kim, P., Yu, J., Lee, H.-M., Kwon, S.-B., Kim, K. P., Lee, S.-H., Park, Tags: Immunology, Microbiology, Virology & Host Pathogen Interaction Articles Source Type: research

Regulation of death receptor signaling by the autophagy protein TP53INP2
TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquit...
Source: EMBO Journal - May 15, 2019 Category: Molecular Biology Authors: Ivanova, S., Polajnar, M., Narbona-Perez, A. J., Hernandez-Alvarez, M. I., Frager, P., Slobodnyuk, K., Plana, N., Nebreda, A. R., Palacin, M., Gomis, R. R., Behrends, C., Zorzano, A. Tags: Cancer, Autophagy & Cell Death Articles Source Type: research

Lining up for quality control: linear ubiquitin and proteotoxicity
The post-translational modification of proteins with linear ubiquitin (Ub) has previously been shown to be important for regulating inflammatory and immunological signaling in response to cellular pathogens. However, other functions of linear Ub are currently poorly defined. In this issue of The EMBO Journal, van Well and colleagues define a new role for linear Ub in regulating the intracellular stability and proteotoxicity of protein aggregates implicated in neurodegenerative diseases. This work provides new insights into the pathologic and potential therapeutic implications for linear Ub in the pathogenesis of these diso...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Wiseman, R. L. Tags: Neuroscience, Post-translational Modifications, Proteolysis & Proteomics News [amp ] Views Source Type: research

A choreography of intracellular Ca2+ and extracellular ATP to refine auditory nociceptors before hearing
Our hearing ability is exquisite in terms of sensitivity, dynamic range, and frequency discrimination. However, this acuity comes at the price of high vulnerability to a range of adverse influences—including sustained loud sounds. One may therefore expect that the ear is equipped with an alert system that can help prevent hearing damage, akin to the nociceptive sensory system that safeguards us from undergoing tissue injury. Here, Ceriani et al reveal a complex signaling interplay of sensory and non-sensory cells that helps setting up such a system in development. (Source: EMBO Journal)
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Leitner, M. G., Oliver, D. Tags: Development & Differentiation, Membrane & Intracellular Transport News [amp ] Views Source Type: research

The assembly of lipid droplets and their roles in challenged cells
(Source: EMBO Journal)
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Henne, W. M., Reese, M. L., Goodman, J. M. Tags: Corrigendum Source Type: research

A supporting ecosystem to mature extracellular vesicles into clinical application
Research into extracellular vesicles (EV) has yielded important biological insights and raised the prospect of developing novel diagnostics and therapeutics for a wide range of pathologies. As with other emerging and transformative fields in research, it will require a broad, supportive base for EV research to mature and to develop clinical application. Here, we identify several focus areas to further improve reproducibility and reliability specifically for EV research and make recommendations for minimal experimental guidelines, transparency tools, reference materials, validation, identification of contaminants, data shar...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: De Wever, O., Hendrix, A. Tags: Membrane & Intracellular Transport Commentary Source Type: research

Binding of IFT22 to the intraflagellar transport complex is essential for flagellum assembly
Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate mo...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Wachter, S., Jung, J., Shafiq, S., Basquin, J., Fort, C., Bastin, P., Lorentzen, E. Tags: Cell Adhesion, Polarity & Cytoskeleton, Membrane & Intracellular Transport, Structural Biology Articles Source Type: research

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes
The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII. We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particula...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Maslon, M. M., Braunschweig, U., Aitken, S., Mann, A. R., Kilanowski, F., Hunter, C. J., Blencowe, B. J., Kornblihtt, A. R., Adams, I. R., Caceres, J. F. Tags: RNA Biology, Transcription Articles Source Type: research

Protein translocation by the SecA ATPase occurs by a power-stroke mechanism
SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Catipovic, M. A., Bauer, B. W., Loparo, J. J., Rapoport, T. A. Tags: Membrane & Intracellular Transport Articles Source Type: research

A protein quality control pathway regulated by linear ubiquitination
Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unw...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: van Well, E. M., Bader, V., Patra, M., Sanchez-Vicente, A., Meschede, J., Furthmann, N., Schnack, C., Blusch, A., Longworth, J., Petrasch-Parwez, E., Mori, K., Arzberger, T., Trümbach, D., Angersbach, L., Showkat, C., Sehr, D. A., Berlemann, L. A. Tags: Neuroscience, Post-translational Modifications, Proteolysis & Proteomics Articles Source Type: research

Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lip...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Dudley, L. J., Cabodevilla, A. G., Makar, A. N., Sztacho, M., Michelberger, T., Marsh, J. A., Houston, D. R., Martens, S., Jiang, X., Gammoh, N. Tags: Autophagy & Cell Death, Membrane & Intracellular Transport Articles Source Type: research

Mutant Lef1 controls Gata6 in sebaceous gland development and cancer
Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a β-catenin-independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9-positive Lef1-negative hair follicle progenitors that give rise to the upper SG. Overexpression of Gata6 by in utero lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutan...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Oules, B., Rognoni, E., Hoste, E., Goss, G., Fiehler, R., Natsuga, K., Quist, S., Mentink, R., Donati, G., Watt, F. M. Tags: Cancer, Development & Differentiation, Transcription Articles Source Type: research

ALYREF links 3'-end processing to nuclear export of non-polyadenylated mRNAs
The RNA-binding protein ALYREF plays key roles in nuclear export and also 3'-end processing of polyadenylated mRNAs, but whether such regulation also extends to non-polyadenylated RNAs is unknown. Replication-dependent (RD)-histone mRNAs are not polyadenylated, but instead end in a stem-loop (SL) structure. Here, we demonstrate that ALYREF prevalently binds a region next to the SL on RD-histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and promotes its recruitment. ALYREF promotes histone pre-mRNA 3'-end processing by facilitating U7-snRNP recruitment through physical interaction with the U7-snRNP-spe...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Fan, J., Wang, K., Du, X., Wang, J., Chen, S., Wang, Y., Shi, M., Zhang, L., Wu, X., Zheng, D., Wang, C., Wang, L., Tian, B., Li, G., Zhou, Y., Cheng, H. Tags: RNA Biology Articles Source Type: research

Coordinated calcium signalling in cochlear sensory and non-sensory cells refines afferent innervation of outer hair cells
Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non-sensory cells of the greater epithelial ridge...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Ceriani, F., Hendry, A., Jeng, J.-Y., Johnson, S. L., Stephani, F., Olt, J., Holley, M. C., Mammano, F., Engel, J., Kros, C. J., Simmons, D. D., Marcotti, W. Tags: Development & Differentiation, Membrane & Intracellular Transport Articles Source Type: research

TRAF6 directs FOXP3 localization and facilitates regulatory T-cell function through K63-linked ubiquitination
Regulatory T cells (Tregs) are crucial mediators of immune control. The characteristic gene expression and suppressive functions of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. Transcriptional regulation of the Foxp3 gene has been studied in depth, but both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. Here, we found that TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 ...
Source: EMBO Journal - April 25, 2019 Category: Molecular Biology Authors: Ni, X., Kou, W., Gu, J., Wei, P., Wu, X., Peng, H., Tao, J., Yan, W., Yang, X., Lebid, A., Park, B. V., Chen, Z., Tian, Y., Fu, J., Newman, S., Wang, X., Shen, H., Li, B., Blazar, B. R., Wang, X., Barbi, J., Pan, F., Lu, L. Tags: Cancer, Immunology, Post-translational Modifications, Proteolysis & Proteomics Articles Source Type: research

Pancreatic development: one cell at a (pseudo)time
Gaining a comprehensive understanding of the principles guiding lineage differentiation during organogenesis is a challenge. In this issue, Yu et al (2019) tackle this venture by taking advantage of single-cell transcriptomic analyses and multiple mouse genetic tools. Their work provides new insights into pancreatic endocrine and exocrine cell differentiation landscapes and identifies new pathways regulating pancreatic lineage allocation in vivo. (Source: EMBO Journal)
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Liu, Z., Sneddon, J. B. Tags: Development & Differentiation, Systems & Computational Biology News [amp ] Views Source Type: research

To Fis or not to Fuse? This is the question!
This study highlights the limitation of categorizing mitochondrial dynamics proteins based on architecture measurements, given the general tendency to associate fragmentation with activation of fission. (Source: EMBO Journal)
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Liesa, M., Van der Bliek, A., Shirihai, O. S. Tags: Membrane & Intracellular Transport News [amp ] Views Source Type: research

SUMOylation promotes protective responses to DNA-protein crosslinks
DNA-protein crosslinks (DPCs) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, the mechanisms by which cells respond to and overcome DPCs remain incompletely understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT-type metalloprotease SPRTN/DVC1, which proteolytically processes DPCs during DNA replication in a ubiquitin-regulated manner. Here, we show that chemically induced and defined enzymatic DPCs trigger potent chromatin SUMOylation responses targeting the crosslinked prote...
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Borgermann, N., Ackermann, L., Schwertman, P., Hendriks, I. A., Thijssen, K., Liu, J. C., Lans, H., Nielsen, M. L., Mailand, N. Tags: DNA Replication, Repair & Recombination, Post-translational Modifications, Proteolysis & Proteomics Articles Source Type: research

Slicing and dicing viruses: antiviral RNA interference in mammals
To protect against the harmful consequences of viral infections, organisms are equipped with sophisticated antiviral mechanisms, including cell-intrinsic means to restrict viral replication and propagation. Plant and invertebrate cells utilise mostly RNA interference (RNAi), an RNA-based mechanism, for cell-intrinsic immunity to viruses while vertebrates rely on the protein-based interferon (IFN)-driven innate immune system for the same purpose. The RNAi machinery is conserved in vertebrate cells, yet whether antiviral RNAi is still active in mammals and functionally relevant to mammalian antiviral defence is intensely deb...
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Maillard, P. V., van der Veen, A. G., Poirier, E. Z., Reis e Sousa, C. Tags: Immunology, RNA Biology Review Source Type: research

Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion
The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome–lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affec...
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Baba, T., Toth, D. J., Sengupta, N., Kim, Y. J., Balla, T. Tags: Autophagy & Cell Death, Membrane & Intracellular Transport Articles Source Type: research

Defining multistep cell fate decision pathways during pancreatic development at single-cell resolution
This study provides key insights into the regulatory mechanisms underlying cell fate choice and stepwise cell fate commitment and can be used as a resource to guide the induction of functional islet lineage cells from stem cells in vitro. (Source: EMBO Journal)
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Yu, X.-X., Qiu, W.-L., Yang, L., Zhang, Y., He, M.-Y., Li, L.-C., Xu, C.-R. Tags: Development & Differentiation, Methods & Resources, Systems & Computational Biology Source Type: research

Cideb controls sterol-regulated ER export of SREBP/SCAP by promoting cargo loading at ER exit sites
SREBPs are master regulators of lipid homeostasis and undergo sterol-regulated export from ER to Golgi apparatus for processing and activation via COPII-coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet-associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP-Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb b...
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Su, L., Zhou, L., Chen, F.-J., Wang, H., Qian, H., Sheng, Y., Zhu, Y., Yu, H., Gong, X., Cai, L., Yang, X., Xu, L., Zhao, T.-J., Li, J. Z., Chen, X.-W., Li, P. Tags: Membrane & Intracellular Transport, Metabolism Articles Source Type: research

A spatiotemporally regulated transcriptional complex underlies heteroblastic development of leaf hairs in Arabidopsis thaliana
Heteroblasty refers to a phenomenon that a plant produces morphologically or functionally different lateral organs in an age-dependent manner. In the model plant Arabidopsis thaliana, the production of trichomes (epidermal leaf hairs) on the abaxial (lower) side of leaves is a heteroblastic mark for the juvenile-to-adult transition. Here, we show that the heteroblastic development of abaxial trichomes is regulated by a spatiotemporally regulated complex comprising the leaf abaxial fate determinant (KAN1) and the developmental timer (miR172-targeted AP2-like proteins). We provide evidence that a short-distance chromatin loo...
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Wang, L., Zhou, C.-M., Mai, Y.-X., Li, L.-Z., Gao, J., Shang, G.-D., Lian, H., Han, L., Zhang, T.-Q., Tang, H.-B., Ren, H., Wang, F.-X., Wu, L.-Y., Liu, X.-L., Wang, C.-S., Chen, E.-W., Zhang, X.-N., Liu, C., Wang, J.-W. Tags: Development & Differentiation, Plant Biology, Transcription Articles Source Type: research

The long noncoding RNA ROCKI regulates inflammatory gene expression
Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus. We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and I...
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Zhang, Q., Chao, T.-C., Patil, V. S., Qin, Y., Tiwari, S. K., Chiou, J., Dobin, A., Tsai, C.-M., Li, Z., Dang, J., Gupta, S., Urdahl, K., Nizet, V., Gingeras, T. R., Gaulton, K. J., Rana, T. M. Tags: Immunology, Microbiology, Virology & Host Pathogen Interaction, RNA Biology Articles Source Type: research

Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery
Mitochondrial dynamics is important for life. At center stage for mitochondrial dynamics, the balance between mitochondrial fission and fusion is a set of dynamin-related GTPases that drive mitochondrial fission and fusion. Fission is executed by the GTPases Drp1 and Dyn2, whereas the GTPases Mfn1, Mfn2, and OPA1 promote fusion. Recruitment of Drp1 to mitochondria is a critical step in fission. In yeast, Fis1p recruits the Drp1 homolog Dnm1p to mitochondria through Mdv1p and Caf4p, but whether human Fis1 (hFis1) promotes fission through a similar mechanism as in yeast is not established. Here, we show that hFis1-mediated m...
Source: EMBO Journal - April 15, 2019 Category: Molecular Biology Authors: Yu, R., Jin, S.-B., Lendahl, U., Nister, M., Zhao, J. Tags: Membrane & Intracellular Transport Articles Source Type: research

Bub1--the zombie protein that CRISPR cannot kill
The conserved Bub1 kinase was proposed to be non-essential for the mitotic spindle assembly checkpoint based on recent findings that knocking-out Bub1 by CRISPR/Cas9 did not impair checkpoint function in human cells. New studies now demonstrate that CRISPR/Cas9 Bub1 knockout cells still express low levels of Bub1 protein and that only removal of this remaining Bub1 pool by RNA interference substantially weakens spindle assembly checkpoint signaling. (Source: EMBO Journal)
Source: EMBO Journal - April 1, 2019 Category: Molecular Biology Authors: Meraldi, P. Tags: Cell Cycle, Chromatin, Epigenetics, Genomics & Functional Genomics, Methods & Resources News [amp ] Views Source Type: research

Revealing chromatin organization in metaphase chromosomes
The local structural organization of chromatin in mitotic chromosomes is not well understood. A new cryo-electron tomography study from the Daban laboratory (Chicano et al, 2019) reveals that mitotic chromatin isolated from human cells can assume a plate-like fine structure, containing layers of interdigitated nucleosomes. Such a multilayered organization suggests a possible model for mitotic chromatin compaction. (Source: EMBO Journal)
Source: EMBO Journal - April 1, 2019 Category: Molecular Biology Authors: Fierz, B. Tags: Chromatin, Epigenetics, Genomics & Functional Genomics, Structural Biology News [amp ] Views Source Type: research

Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
Small RNAs post-transcriptionally regulate many processes in bacteria. Base-pairing of sRNAs near ribosome-binding sites in mRNAs inhibits translation, often requiring the RNA chaperone Hfq. In the canonical model, Hfq simultaneously binds sRNAs and mRNA targets to accelerate pairing. Here, we show that the Escherichia coli sRNAs OmrA and OmrB inhibit translation of the diguanylate cyclase DgcM (previously: YdaM), a player in biofilm regulation. In OmrA/B repression of dgcM, Hfq is not required as an RNA interaction platform, but rather unfolds an inhibitory RNA structure that impedes OmrA/B binding. This restructuring inv...
Source: EMBO Journal - April 1, 2019 Category: Molecular Biology Authors: Hoekzema, M., Romilly, C., Holmqvist, E., Wagner, E. G. H. Tags: Microbiology, Virology & Host Pathogen Interaction, RNA Biology Articles Source Type: research

NBS1 promotes the endonuclease activity of the MRE11-RAD50 complex by sensing CtIP phosphorylation
DNA end resection initiates DNA double-strand break repair by homologous recombination. MRE11-RAD50-NBS1 and phosphorylated CtIP perform the first resection step via MRE11-catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11-RAD50 nuclease through direct physical interactions with MRE11. In ...
Source: EMBO Journal - April 1, 2019 Category: Molecular Biology Authors: Anand, R., Jasrotia, A., Bundschuh, D., Howard, S. M., Ranjha, L., Stucki, M., Cejka, P. Tags: DNA Replication, Repair & Recombination Articles Source Type: research

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex
In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1-Bub1 complex. (Source: EMBO Journal)
Source: EMBO Journal - April 1, 2019 Category: Molecular Biology Authors: Zhang, G., Kruse, T., Guasch Boldu, C., Garvanska, D. H., Coscia, F., Mann, M., Barisic, M., Nilsson, J. Tags: Cell Cycle Articles Source Type: research