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New and Prospective Roles for lncRNAs in Organelle Formation and Function.

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New and Prospective Roles for lncRNAs in Organelle Formation and Function.

Trends Genet. 2018 Jul 14;:

Authors: Krause HM

Abstract
The observation that long noncoding RNAs (lncRNAs) represent the majority of transcripts in humans has led to a rapid increase in interest and study. Most of this interest has focused on their roles in the nucleus. However, increasing evidence is beginning to reveal even more functions outside the nucleus, and even outside cells. Many of these roles are mediated by newly discovered properties, including the ability of lncRNAs to interact with lipids, membranes, and disordered protein domains, and to form differentially soluble RNA-protein sub-organelles. This review explores the possibilities enabled by these new properties and abilities, such as likely roles in exosome formation and function.

PMID: 30017312 [PubMed - as supplied by publisher]



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A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A.

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A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A.

Genetics. 2018 Jul 16;:

Authors: Ciftci-Yilmaz S, Au WC, Mishra PK, Eisenstatt JR, Chang J, Dawson AR, Zhu I, Rahman M, Bilke S, Costanzo M, Baryshnikova A, Myers CL, Meltzer PS, Landsman D, Baker RE, Boone C, Basrai MA

Abstract
Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A leads to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide Synthetic Genetic Array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to non-centromeric regions for genome stability. Hir2 interacts with Cse4 invivo and hir2Δ strains exhibit defects in Cse4 proteolysis, and stabilization of chromatin-bound Cse4. Mislocalization of Cse4 to non-centromeric regions with a preferential enrichment at promoter regions was observed in hir2Δ strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1 and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2Δ strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4 thereby promoting genome stability.

PMID: 30012561 [PubMed - as supplied by publisher]



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Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress.

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Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress.

Diseases. 2018 Jul 13;6(3):

Authors: Han Z, Schwoerer MP, Hicks P, Liang J, Ruthel G, Berry CT, Freedman BD, Sagum CA, Bedford MT, Sidhu SS, Sudol M, Harty RN

Abstract
Lassa fever virus (LFV) belongs to the Arenaviridae family and can cause acute hemorrhagic fever in humans. The LFV Z protein plays a central role in virion assembly and egress, such that independent expression of LFV Z leads to the production of virus-like particles (VLPs) that mimic egress of infectious virus. LFV Z contains both PTAP and PPPY L-domain motifs that are known to recruit host proteins that are important for mediating efficient virus egress and spread. The viral PPPY motif is known to interact with specific host WW-domain bearing proteins. Here we identified host WW-domain bearing protein BCL2 Associated Athanogene 3 (BAG3) as a LFV Z PPPY interactor using our proline-rich reading array of WW-domain containing mammalian proteins. BAG3 is a stress-induced molecular co-chaperone that functions to regulate cellular protein homeostasis and cell survival via Chaperone-Assisted Selective Autophagy (CASA). Similar to our previously published findings for the VP40 proteins of Ebola and Marburg viruses, our results using VLP budding assays, BAG3 knockout cells, and confocal microscopy indicate that BAG3 is a WW-domain interactor that negatively regulates egress of LFV Z VLPs, rather than promoting VLP release. Our results suggest that CASA and specifically BAG3 may represent a novel host defense mechanism, whereby BAG3 may dampen egress of several hemorrhagic fever viruses by interacting and interfering with the budding function of viral PPxY-containing matrix proteins.

PMID: 30011814 [PubMed]



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The impact of oncogenic EGFRvIII on the proteome of extracellular vesicles released from glioblastoma cells.

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The impact of oncogenic EGFRvIII on the proteome of extracellular vesicles released from glioblastoma cells.

Mol Cell Proteomics. 2018 Jul 13;:

Authors: Choi D, Montermini L, Kim DK, Meehan B, Roth FP, Rak J

Abstract
Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and down-regulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins. Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also co-localized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.

PMID: 30006486 [PubMed - as supplied by publisher]



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Functional genetic discovery of enzymes using full-scan mass spectrometry metabolomics.

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Functional genetic discovery of enzymes using full-scan mass spectrometry metabolomics.

Biochem Cell Biol. 2018 Jul 12;:

Authors: Caudy AA, Hanchard JA, Hsieh A, Shaan S, Rosebrock AP

Abstract
Our understanding of metabolic networks is incomplete, and new enzymatic activities await discovery in well studied organisms. Mass spectrometric methods for measuring cellular metabolism reveal compounds inside cells that are unexplained by existing maps of metabolic reactions. Current computational models are unable to account for all activities and contents observed within cells. Additional large-scale genetic and biochemical approaches are required to elucidate metabolic gene function. We have used full-scan mass spectrometry metabolomics to examine deletions of candidate enzymes in the model budding yeast Saccharomyces cerevisiae and report the identification of twenty-five candidates that alter metabolite levels. Triumphs and pitfalls of metabolic phenotyping screens are discussed, including estimates of the frequency of uncharacterized eukaryotic genes affecting metabolism and key issues to consider when searching for new enzymatic functions in other organisms.

PMID: 30001498 [PubMed - as supplied by publisher]



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Author Correction: Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

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Author Correction: Genome-wide changes in lncRNA, splicing, and regional gene expression patterns in autism.

Nature. 2018 Jul 11;:

Authors: Parikshak NN, Swarup V, Belgard TG, Irimia M, Ramaswami G, Gandal MJ, Hartl C, Leppa V, de la Torre Ubieta L, Huang J, Lowe JK, Blencowe BJ, Horvath S, Geschwind DH

Abstract
Change history: In this Letter, the labels for splicing events A3SS and A5SS were swapped in column D of Supplementary Table 3a and b. This has been corrected online.

PMID: 29995847 [PubMed - as supplied by publisher]



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The Cytoscape Automation app article collection.

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The Cytoscape Automation app article collection.

F1000Res. 2018;7:800

Authors: Demchak B, Otasek D, Pico AR, Bader GD, Ono K, Settle B, Sage E, Morris JH, Longabaugh W, Lopes C, Kucera M, Treister A, Schwikowski B, Molenaar P, Ideker T

Abstract
Cytoscape is the premiere platform for interactive analysis, integration and visualization of network data. While Cytoscape itself delivers much basic functionality, it relies on community-written apps to deliver specialized functions and analyses. To date, Cytoscape's CyREST feature has allowed researchers to write workflows that call basic Cytoscape functions, but provides no access to its high value app-based functions. With Cytoscape Automation, workflows can now call apps that have been upgraded to expose their functionality. This article collection is a resource to assist readers in quickly and economically leveraging such apps in reproducible workflows that scale independently to large data sets and production runs.

PMID: 29983926 [PubMed]



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CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

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CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

Nature. 2018 Jul 04;:

Authors: Zimmermann M, Murina O, Reijns MAM, Agathanggelou A, Challis R, Tarnauskaitė Ž, Muir M, Fluteau A, Aregger M, McEwan A, Yuan W, Clarke M, Lambros MB, Paneesha S, Moss P, Chandrashekhar M, Angers S, Moffat J, Brunton VG, Hart T, de Bono J, Stankovic T, Jackson AP, Durocher D

Abstract
The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.

PMID: 29973717 [PubMed - as supplied by publisher]



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COSSMO: predicting competitive alternative splice site selection using deep learning.

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COSSMO: predicting competitive alternative splice site selection using deep learning.

Bioinformatics. 2018 Jul 01;34(13):i429-i437

Authors: Bretschneider H, Gandhi S, Deshwar AG, Zuberi K, Frey BJ

Abstract
Motivation: Alternative splice site selection is inherently competitive and the probability of a given splice site to be used also depends on the strength of neighboring sites. Here, we present a new model named the competitive splice site model (COSSMO), which explicitly accounts for these competitive effects and predicts the percent selected index (PSI) distribution over any number of putative splice sites. We model an alternative splicing event as the choice of a 3' acceptor site conditional on a fixed upstream 5' donor site or the choice of a 5' donor site conditional on a fixed 3' acceptor site. We build four different architectures that use convolutional layers, communication layers, long short-term memory and residual networks, respectively, to learn relevant motifs from sequence alone. We also construct a new dataset from genome annotations and RNA-Seq read data that we use to train our model.
Results: COSSMO is able to predict the most frequently used splice site with an accuracy of 70% on unseen test data, and achieve an R2 of 0.6 in modeling the PSI distribution. We visualize the motifs that COSSMO learns from sequence and show that COSSMO recognizes the consensus splice site sequences and many known splicing factors with high specificity.
Availability and implementation: Model predictions, our training dataset, and code are available from http://cossmo.genes.toronto.edu.
Supplementary information: Supplementary data are available at Bioinformatics online.

PMID: 29949959 [PubMed - in process]



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Co-targeting ephrin receptor tyrosine kinases A2 and A3 in cancer stem cells reduces growth of recurrent glioblastoma.

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Co-targeting ephrin receptor tyrosine kinases A2 and A3 in cancer stem cells reduces growth of recurrent glioblastoma.

Cancer Res. 2018 Jun 26;:

Authors: Qazi MA, Vora P, Venugopal C, Adams J, Singh M, Hu AX, Gorelik M, Subapanditha MK, Savage N, Yang J, Chokshi C, London M, Gont A, Bobrowski D, Grinshtein N, Brown KR, Murty NK, Nilvebrant J, Kaplan DR, Moffat J, Sidhu S, Singh SK

Abstract
Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by glioblastoma stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphR that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (Cytometry by Time-Of-Flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSC to identify putative rGBM-specific EphR. EphA2 and EphA3 co-expression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EphA2 and EphA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo. Treatment of rGBM with a bispecific antibody (BsAb) against EphA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of Akt and Erk and increased cellular differentiation. In conclusion, we show that EphA2 and EphA3 together mark a GSC population in rGBM and that strategic co-targeting of EphA2 and EphA3 presents a novel and rational therapeutic approach for rGBM.

PMID: 29945963 [PubMed - as supplied by publisher]



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