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Nucleolar RNA polymerase II drives ribosome biogenesis.

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Nucleolar RNA polymerase II drives ribosome biogenesis.

Nature. 2020 09;585(7824):298-302

Authors: Abraham KJ, Khosraviani N, Chan JNY, Gorthi A, Samman A, Zhao DY, Wang M, Bokros M, Vidya E, Ostrowski LA, Oshidari R, Pietrobon V, Patel PS, Algouneh A, Singhania R, Liu Y, Yerlici VT, De Carvalho DD, Ohh M, Dickson BC, Hakem R, Greenblatt JF, Lee S, Bishop AJR, Mekhail K

Abstract
Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

PMID: 32669707 [PubMed - indexed for MEDLINE]



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Mapping signalling perturbations in myocardial fibrosis via the integrative phosphoproteomic profiling of tissue from diverse sources.

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Mapping signalling perturbations in myocardial fibrosis via the integrative phosphoproteomic profiling of tissue from diverse sources.

Nat Biomed Eng. 2020 09;4(9):889-900

Authors: Kuzmanov U, Wang EY, Vanderlaan R, Kim DH, Lee SH, Hadipour-Lakmehsari S, Guo H, Zhao Y, McFadden M, Sharma P, Billia F, Radisic M, Gramolini A, Emili A

Abstract
Study of the molecular basis of myocardial fibrosis is hampered by limited access to tissues from human patients and by confounding variables associated with sample accessibility, collection, processing and storage. Here, we report an integrative strategy based on mass spectrometry for the phosphoproteomic profiling of normal and fibrotic cardiac tissue obtained from surgical explants from patients with hypertrophic cardiomyopathy, from a transaortic-constriction mouse model of cardiac hypertrophy and fibrosis, and from a heart-on-a-chip model of cardiac fibrosis. We used the integrative approach to map the relative abundance of thousands of proteins, phosphoproteins and phosphorylation sites specific to each tissue source, to identify key signalling pathways driving fibrosis and to screen for anti-fibrotic compounds targeting glycogen synthase kinase 3, which has a consistent role as a key mediator of fibrosis in all three types of tissue specimen. The integrative disease-modelling strategy may reveal new insights into mechanisms of cardiac disease and serve as a test bed for drug screening.

PMID: 32661320 [PubMed - indexed for MEDLINE]



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A Genetic Map of the Response to DNA Damage in Human Cells.

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A Genetic Map of the Response to DNA Damage in Human Cells.

Cell. 2020 Jul 09;:

Authors: Olivieri M, Cho T, Álvarez-Quilón A, Li K, Schellenberg MJ, Zimmermann M, Hustedt N, Rossi SE, Adam S, Melo H, Heijink AM, Sastre-Moreno G, Moatti N, Szilard RK, McEwan A, Ling AK, Serrano-Benitez A, Ubhi T, Feng S, Pawling J, Delgado-Sainz I, Ferguson MW, Dennis JW, Brown GW, Cortés-Ledesma F, Williams RS, Martin A, Xu D, Durocher D

Abstract
The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

PMID: 32649862 [PubMed - as supplied by publisher]



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Author Correction: In-cell identification and measurement of RNA-protein interactions.

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Author Correction: In-cell identification and measurement of RNA-protein interactions.

Nat Commun. 2020 Jul 08;11(1):3498

Authors: Graindorge A, Pinheiro I, Nawrocka A, Mallory AC, Tsvetkov P, Gil N, Carolis C, Buchholz F, Ulitsky I, Heard E, Taipale M, Shkumatava A

Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PMID: 32641823 [PubMed - in process]



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Protein Interactions of the Mechanosensory Proteins Wsc2 and Wsc3 for Stress Resistance in Saccharomyces cerevisiae.

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Protein Interactions of the Mechanosensory Proteins Wsc2 and Wsc3 for Stress Resistance in Saccharomyces cerevisiae.

G3 (Bethesda). 2020 Jul 08;:

Authors: Vélez-Segarra V, González-Crespo S, Santiago-Cartagena E, Vázquez-Quiñones LE, Martínez-Matías N, Otero Y, Zayas JJ, Siaca R, Del Rosario J, Mejías I, Aponte JA, Collazo NC, Lasso FJ, Snider J, Jessulat M, Aoki H, Rymond BC, Babu M, Stagljar I, Rodríguez-Medina JR

Abstract
Antifungal drug discovery and design is very challenging because of the considerable similarities in genetic features and metabolic pathways between fungi and humans. However, cell wall composition represents a notable point of divergence. Therefore, a research strategy was designed to improve our understanding of the mechanisms for maintaining fungal cell wall integrity, and to identify potential targets for new drugs that modulate the underlying protein-protein interactions in Saccharomyces cerevisiae This study defines roles for Wsc2p and Wsc3p and their interacting protein partners in the cell wall integrity signaling and cell survival mechanisms that respond to treatments with fluconazole and hydrogen peroxide. By combined genetic and biochemical approaches, we report the discovery of 12 novel protein interactors of Wsc2p and Wsc3p. Of these, Wsc2p interacting partners Gtt1p and Yck2p, have opposing roles in the resistance and sensitivity to fluconazole treatments respectively. The interaction of Wsc2p with Ras2p was confirmed by iMYTH and IP-MS approaches and is shown to play a dominant role in response to oxidative stress induced by hydrogen peroxide. Consistent with an earlier study, Ras2p was also identified as an interacting partner of Wsc1p and Mid2p cell wall integrity signaling proteins. Collectively, this study expands the interaction networks of the mechanosensory proteins of the Cell Wall Integrity pathway.

PMID: 32641451 [PubMed - as supplied by publisher]



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Generation of Functional Liver Sinusoidal Endothelial Cells from Human Pluripotent Stem-Cell-Derived Venous Angioblasts.

Generation of Functional Liver Sinusoidal Endothelial Cells from Human Pluripotent Stem-Cell-Derived Venous Angioblasts.

Cell Stem Cell. 2020 Jul 01;:

Authors: Gage BK, Liu JC, Innes BT, MacParland SA, McGilvray ID, Bader GD, Keller GM

Abstract
Liver sinusoidal endothelial cells (LSECs) form a highly specialized microvasculature that plays a critical role in liver function and disease. To better understand this role, we developed a strategy to generate LSECs from human pluripotent stem cells (hPSCs) by first optimizing the specification of arterial and venous angioblasts and derivative endothelial populations. Induction of a LSEC-like fate by hypoxia, cyclic AMP (cAMP) agonism, and transforming growth factor β (TGF-β) inhibition revealed that venous endothelial cells responded more rapidly and robustly than the arterial cells to upregulate LSEC markers and functions in vitro. Upon intrahepatic transplantation in neonates, venous angioblasts engrafted the liver and generated mature, fenestrated LSECs with scavenger functions and molecular profiles of primary human LSECs. When transplanted into the liver of adult mice, angioblasts efficiently gave rise to mature LSECs with robust factor VIII (FVIII) production. Humanization of the murine liver with hPSC-derived LSECs provides a tractable system for studying the biology of this key liver cell type.

PMID: 32640183 [PubMed - as supplied by publisher]



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In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization.

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In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization.

Front Cell Dev Biol. 2020;8:489

Authors: Rocha LA, Gomes ED, Afonso JL, Granja S, Baltazar F, Silva NA, Shoichet MS, Sousa RA, Learmonth DA, Salgado AJ

Abstract
Vascular disruption following spinal cord injury (SCI) decisively contributes to the poor functional recovery prognosis facing patients with the condition. Using a previously developed gellan gum hydrogel to which the adhesion motif GRGDS was grafted (GG-GRGDS), this work aimed to understand the ability of adipose-derived stem cells (ASCs) to impact vascular organization of human umbilical vein endothelial cells (HUVECs), and how this in turn affects neurite outgrowth of dorsal root ganglia (DRG) explants. Our data shows that culturing these cells together lead to a synergistic effect as showed by increased stimulation of neuritogenesis on DRG. Importantly, HUVECs were only able to assemble into vascular-like structures when cultured in the presence of ASCs, which shows the capacity of these cells in reorganizing the vascular milieu. Analysis of selected neuroregulatory molecules showed that the co-culture upregulated the secretion of several neurotrophic factors. On the other hand, ASCs, and ASCs + HUVECs presented a similar profile regarding the presence of angiotrophic molecules herein analyzed. Finally, the implantation of GG-GRGDS hydrogels encapsulating ASCs in the chick chorioallantoic membrane (CAM) lead to increases in vascular recruitment toward the hydrogels in comparison to GG-GRGDS alone. This indicates that the combination of ASCs with GG-GRGDS hydrogels could promote re-vascularization in trauma-related injuries in the central nervous system and thus control disease progression and induce functional recovery.

PMID: 32612997 [PubMed]



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Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

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Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Cell. 2020 Jun 23;:

Authors: Lee H, Beilhartz GL, Kucharska I, Raman S, Cui H, Lam MHY, Liang H, Rubinstein JL, Schramek D, Julien JP, Melnyk RA, Taipale M

Abstract
Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

PMID: 32589945 [PubMed - as supplied by publisher]



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Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

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Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

Science. 2020 06 26;368(6498):

Authors: Kuzmin E, VanderSluis B, Nguyen Ba AN, Wang W, Koch EN, Usaj M, Khmelinskii A, Usaj MM, van Leeuwen J, Kraus O, Tresenrider A, Pryszlak M, Hu MC, Varriano B, Costanzo M, Knop M, Moses A, Myers CL, Andrews BJ, Boone C

Abstract
Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event. Mapping of digenic interactions for a deletion mutant of each paralog, and of trigenic interactions for the double mutant, provides insight into their roles and a quantitative measure of their functional redundancy. Trigenic interaction analysis distinguishes two classes of paralogs: a more functionally divergent subset and another that retained more functional overlap. Gene feature analysis and modeling suggest that evolutionary trajectories of duplicated genes are dictated by combined functional and structural entanglement factors.

PMID: 32586993 [PubMed - indexed for MEDLINE]



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Functional genomics identifies new synergistic therapies for retinoblastoma.

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Functional genomics identifies new synergistic therapies for retinoblastoma.

Oncogene. 2020 07;39(31):5338-5357

Authors: Aubry A, Pearson JD, Huang K, Livne-Bar I, Ahmad M, Jagadeesan M, Khetan V, Ketela T, Brown KR, Yu T, Lu S, Wrana JL, Moffat J, Bremner R

Abstract
Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily with RB1 loss or, rarely, MYCN amplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens in RB1null and RB1wt;MYCNamp orthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53-BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53-p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53-BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.

PMID: 32572160 [PubMed - indexed for MEDLINE]



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