DOCSLIB.ORG

DDX17 Promotes the Production of Infectious HIV-1 Particles Through Modulating Viral RNA Packaging and Translation Frameshift

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
DDX17 Promotes the Production of Infectious HIV-1 Particles Through Modulating Viral RNA Packaging and Translation Frameshift Virology 443 (2013) 384–392 Contents lists available at SciVerse ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro DDX17 promotes the production of infectious HIV-1 particles through modulating viral RNA packaging and translation frameshift René-Pierre Lorgeoux a,c, Qinghua Pan a, Yann Le Duff a,b, Chen Liang a,b,c,n a Lady Davis Institute-Jewish General Hospital, Montreal, QC, Canada H3T 1E2 b Departments of Medicine and Microbiology & Immunology, McGill University, Montreal, QC, Canada H3A 2B4 c Microbiology & Immunology, McGill University, Montreal, QC, Canada H3A 2B4 article info abstract Article history: RNA helicases are a large family of proteins that rearrange RNA structures and remodel ribonucleic Received 21 February 2013 protein complexes using energy derived from hydrolysis of nucleotide triphosphates. They have been Returned to author for revisions shown to participate in every step of RNA metabolism. In the past decade, an increasing number of 24 March 2013 helicases were shown to promote or inhibit the replication of different viruses, including human Accepted 18 May 2013 immunodeficiency virus type 1. Among these helicases, the DEAD-box RNA helicase DDX17 was recently Available online 12 June 2013 reported to modulate HIV-1 RNA stability and export. In this study, we further show that the helicase Keywords: activity of DDX17 is required for the production of infectious HIV-1 particles. Over expression of the HIV DDX17 mutant DQAD in HEK293 cells reduces the amount of packaged viral genomic RNA and DDX17 diminishes HIV-1 Gag-Pol frameshift. Altogether, these data demonstrate that DDX17 promotes the Helicase production of HIV-1 infectious particles by modulating HIV-1 RNA metabolism. RNA packaging & Infectivity 2013 Elsevier Inc. All rights reserved. Introduction to promote or inhibit HIV-1 replication at distinct steps. These include RNA helicase A (RHA), Moloney leukemia virus 10 (MOV10), RNA helicases are a large family of proteins that are involved in XPB/XPD, Ku70/Ku80, Werner syndrome (WRN) helicase, DDX1, virtually all the steps of RNA metabolism (Cordin et al., 2006; DDX3, DDX17, DDX24, Upf1 and DHX30 (reviewed in (Jeang and Linder and Jankowsky, 2011). They utilize the energy derived from Yedavalli, 2006; Lorgeoux et al., 2012; Ranji and Boris-Lawrie, 2010; nucleotide triphosphates (NTPs) hydrolysis to unwind nucleic Steimer and Klostermeier, 2012)). RHA promotes HIV-1 reverse acids and remodel ribonucleic protein (RNP) complexes (Tanner transcription, transcription and translation (Bolinger et al., 2010; and Linder, 2001). RNA helicases are classified into five super Fujii et al., 2001; Roy et al., 2006; Xing et al., 2011). MOV10 inhibits families (SF1 to SF5) on the basis of their conserved motifs. They viral reverse transcription (Burdick et al., 2010; Furtak et al., 2010). are prevalent in eukaryotes, prokaryotes and viruses (Kwong et al., DDX1, DDX3 and DDX17 promote Rev-dependent HIV-1 RNA export 2005; Singleton et al., 2007). The SF2 has the most members, and (Fang et al., 2004; Naji et al., 2012; Yedavalli et al., 2004). In addition includes the DEAD-box helicases that harbor the Asp-Glu-Ala-Asp to its role in HIV-1 RNA export, DDX3 also enhances HIV-1 signature motif (Linder et al., 1989). In humans, malfunction of translation (Geissler et al., 2012; Lai et al., 2010, 2008; Soto-Rifo helicases causes various types of cancers (Clark et al., 2008; Fuller- et al., 2012, 2013). Upf1 and DDX17 are involved in HIV-1 RNA Pace and Moore, 2011; Rezazadeh, 2012). degradation (Ajamian et al., 2008; Zhu et al., 2011). DDX24 Viruses need helicases to successfully replicate their nucleic promotes HIV-1 RNA packaging (Ma et al., 2008), while DHX30 acid genome. Some viruses carry their own helicase, such as was reported to inhibit this step (Zhou et al., 2008). Recently, Hepatitis C virus that encodes the NS3 protein (Kanai et al., SLFN11 was shown to inhibit HIV-1 translation in a codon-usage- 1995; Kim et al., 1995), while others need to hijack cellular ones dependent manner (Li et al., 2012), adding a new layer of viral RNA to assist viral replication. Human immunodeficiency virus type 1 regulation by cellular helicases. (HIV-1), like all retroviruses, does not encode a viral helicase. Over DDX17, also known as RH70, has two isoforms, p72 and p82, as the past decade, many cellular RNA helicases have been reported a result of alternative translation of its messenger RNA (mRNA) (Uhlmann-Schiffler et al., 2002). DDX17 can form heterodimers with its paralog DDX5 (Ogilvie et al., 2003) and is involved in n Corresponding author at: Jewish General Hospital Lady Davis Institute McGill multiple aspects of RNA metabolism. Through interaction with AIDS Centre, Rm 326 3755 Cote Ste-Catherine Road Montreal, QC, Canada H3T 1E2. Fax: +1 514 3407535. other cellular factors, including HDAC1 (Wilson et al., 2004), E-mail address: [email protected] (C. Liang). estrogen receptor alpha (Wortham et al., 2009) and U1snRNP 0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.05.026 R.-P. Lorgeoux et al. / Virology 443 (2013) 384–392 385 (Lee, 2002), DDX17 regulates gene transcription and alternative with 10% Fetal Bovine Serum (FBS). Plasmids and short interfering splicing. Its dysfunction can lead to cancer development RNA (siRNA) were transfected into cells using lipofectamine2000 (Dardenne et al., 2012; Honig et al., 2002; Shin et al., 2007). according to the manufacturer's protocol. Recently, DDX17 was reported to act as a co-factor of the zinc fi nger antiviral protein ZAP (Chen et al., 2008), which is involved Knockdown of DDX5 and DDX17 in HeLa cells in the degradation of the multiply spliced HIV-1 mRNA (Zhu et al., 2011). Another study by Naji et al., (2012) showed that DDX17 siRNA oligonucleotides targeting DDX17 (nt 1694–1713) were associates with HIV-1 Rev protein, and promotes the nuclear purchased from Qiagen (5′-AAT ACA CCT ATG GTC AAG GCA-3′) export of HIV-1 transcripts . We now further show that DDX17 (Cat. no. 1027423). siRNAs oligonucleotides targeting DDX5 and affects the production of infectious HIV-1 particles. Knockdown the DDX5-DDX17 subfamily were previously described (Jalal et al., and over expression of DDX17 change the ratios of unspliced vs 2007). Control siRNA was purchased from Qiagen (Cat. No. spliced HIV-1 RNA. Furthermore, over expression of the DDX17 1027281). shRNA targeting DDX17 (nt 1525–1545) was purchased DQAD mutant leads to a dramatic decrease in the amount of from SIGMA (5′-CCC AAT CTG ATG TAT CAG GAT-3′). Control shRNA fi packaged viral RNA. Moreover, DDX17 is required for ef cient Gag was also purchased form SIGMA (Cat. No. SHC016). HeLa cells processing through its effect on HIV-1 Gag-Pol frameshift. Alto- (2 Â 105 per well) were seeded in 6-well plates 1 day before siRNA gether, our results demonstrate new roles for DDX17 in promoting transfection. DDX5 and DDX17 were either knocked down indivi- the production of infectious HIV-1 particles through regulating dually, or simultaneously. 10 nM of each siRNA was used in HIV-1 Gag-Pol frameshift and genomic RNA packaging. transfection. After two sequential siRNA transfections, the cells were transfected with HIV-1 NL4-3 DNA. Cells and supernatants were harvested 40 h post HIV-1 NL4-3 transfection. Protein levels Materials and methods were analyzed by Western blotting, virus production was assessed by infecting the TZM-bl indicator cells. Plasmid DNA, viruses and antibodies The cDNA clone of DDX17 gene was purchased from ATCC DDX17 overexpression in HEK293 cells (MGC-2030). Short and long isoforms of DDX17 cDNA were 5 amplified by PCR and a Flag tag was added at the N terminus using HEK293 cells (5 Â 10 per well) were seeded in 6-well plates primer pairs DDX17-SS/DDX17-A, DDX17-SL/DDX17-A, respectively 1 day before transfection. 500 ng of DDX17 DNA constructs were (Table 1). The PCR products were digested with BamHI and MulI and co-transfected with 100 ng Tet-ON plasmid and 100 ng HIV-1 NL4-3. inserted into the pRetroX-Tight-Pur retroviral vector (Clontech) to Cells were washed in 1 Â phosphate buffered saline the next day and create DNA plasmids Tet-DDX17S and Tet-DDX17L that are expressed fresh DMEM containing 100 ng/mL doxycyclin was added to induce in response to doxycyclin. DDX17 mutants were generated by site- DDX17 expression. Cells and supernatants were harvested 40 h post directed mutagenesis using primers DQAD-S and DQAD-A (Table 1). HIV-1 NL4-3 transfection. Protein levels were analyzed by Western DDX5 cDNA clone was kindly provided by Dr Stahl (Rossler et al., blotting and virus production was measured by infecting TZM-bl 2000). The infectious HIV-1 proviral DNA clone NL4-3 was obtained indicator cells. from the NIH AIDS Research and Reference Reagent Program. Tat plasmid was kindly provided by Dr. Gatignol (Gatignol et al., 1991). Viral RNA analysis TheframeshiftluciferasereporterconstructsFS(0)andFS(-1)were previously described (Grentzmann et al., 1998)andprovidedby Levels of viral RNA in cells were assessed by Northern blots as Dr. Brakier-Gingras. HIV-1 viruses were generated by transfecting previously described (Roy et al., 2006; Zhou et al., 2008) (and HEK293T cells with the proviral DNA clone NL4-3, using lipofecta- RT-PCR followed by Southern blot, described below). Briefly, mine 2000 (Invitrogen) according to the manufacturer's instruction. transfected cells were washed with 1 Â phosphate buffered saline When indicated, the vesicular stomatitis virus (VSV) glycoprotein and lysed in Trizol (Invitrogen). RNA was extracted according to (G) was used to pseudotype HIV-1 particles. the manufacturer's instructions. Northern blot analysis was per- Anti-Flag and anti-tubulin antibodies were purchased from Sigma; formed as previously described (Zhou et al., 2008).
Load more

Recommended publications
  • Estrogen Regulation and Physiopathologic Significance of Alternative Promoters in Breast Cancer
    Published OnlineFirst April 20, 2010; DOI: 10.1158/0008-5472.CAN-09-3988 Published OnlineFirst on April 20, 2010 as 10.1158/0008-5472.CAN-09-3988 Tumor and Stem Cell Biology Cancer Research Estrogen Regulation and Physiopathologic Significance of Alternative Promoters in Breast Cancer Martin Dutertre1, Lise Gratadou1, Etienne Dardenne1, Sophie Germann1, Samaan Samaan1, Rosette Lidereau2, Keltouma Driouch2, Pierre de la Grange3, and Didier Auboeuf1 Abstract Alternative promoters (AP) occur in >30% protein-coding genes and contribute to proteome diversity. How- ever, large-scale analyses of AP regulation are lacking, and little is known about their potential physiopatho- logic significance. To better understand the transcriptomic effect of estrogens, which play a major role in breast cancer, we analyzed gene and AP regulation by estradiol in MCF7 cells using pan-genomic exon arrays. We thereby identified novel estrogen-regulated genes (ERG) and determined the regulation of AP-encoded transcripts in 150 regulated genes. In <30% cases, APs were regulated in a similar manner by estradiol, whereas in >70% cases, they were regulated differentially. The patterns of AP regulation correlated with the patterns of estrogen receptor α (ERα) and CCCTC-binding factor (CTCF) binding sites at regulated gene loci. Interest- ingly, among genes with differentially regulated (DR) APs, we identified cases where estradiol regulated APs in an opposite manner, sometimes without affecting global gene expression levels. This promoter switch was mediated by the DDX5/DDX17 family of ERα coregulators. Finally, genes with DR promoters were preferen- tially involved in specific processes (e.g., cell structure and motility, and cell cycle). We show, in particular, that isoforms encoded by the NET1 gene APs, which are inversely regulated by estradiol, play distinct roles in cell adhesion and cell cycle regulation and that their expression is differentially associated with prognosis in ER+ breast cancer.
    [Show full text]
  • DDX17 Specifically, and Independently of DDX5, Controls Use of the HIV A4/5 Splice Acceptor Cluster and Is Essential for Efficient Replication of HIV
    Article DDX17 Specifically, and Independently of DDX5, Controls Use of the HIV A4/5 Splice Acceptor Cluster and Is Essential for Efficient Replication of HIV Nyaradzai Sithole 1, Claire A. Williams 1,†, Aisling M. Vaughan 1,‡, Julia C. Kenyon 1,2 and Andrew M.L. Lever 1,3 1 - Department of Medicine, University of Cambridge, Cambridge, CB2 0QQ, UK 2 - Department of Microbiology and Immunology, National University of Singapore, Singapore 117545 3 - Department of Medicine, National University of Singapore, Singapore 119228 Correspondence to Andrew M.L. Lever: Department of Medicine, University of Cambridge, Cambridge, CB2 0QQ, UK. [email protected] https://doi.org/10.1016/j.jmb.2018.06.052 Edited by Eric O. Freed Abstract HIV splicing involves five splice donor and eight splice acceptor sequences which, together with cryptic splice sites, generate over 100 mRNA species. Ninety percent of both partially spliced and fully spliced transcripts utilize the intrinsically weak A4/A5 3′ splice site cluster. We show that DDX17, but not its close paralog DDX5, specifically controls the usage of this splice acceptor group. In its absence, production of the viral envelope protein and other regulatory and accessory proteins is grossly reduced, while Vif, which uses the A1 splice acceptor, is unaffected. This is associated with a profound decrease in viral export from the cell. Loss of Vpu expression causing upregulation of cellular Tetherin compounds the phenotype. DDX17 utilizes distinct RNA binding motifs for its role in efficient HIV replication, and we identify RNA binding motifs essential for its role, while the Walker A, Walker B (DEAD), Q motif and the glycine doublet motif are all dispensable.
    [Show full text]
  • Identification of Protein Features Encoded by Alternative Exons Using Exon Ontology
    Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Resource Identification of protein features encoded by alternative exons using Exon Ontology Léon-Charles Tranchevent,1 Fabien Aubé,1 Louis Dulaurier,1 Clara Benoit-Pilven,1 Amandine Rey,1 Arnaud Poret,1 Emilie Chautard,2 Hussein Mortada,1 François-Olivier Desmet,1 Fatima Zahra Chakrama,1 Maira Alejandra Moreno-Garcia,1 Evelyne Goillot,3 Stéphane Janczarski,1 Franck Mortreux,1 Cyril F. Bourgeois,1,4 and Didier Auboeuf1,4 1Université Lyon 1, ENS de Lyon, CNRS UMR 5239, INSERM U1210, Laboratory of Biology and Modelling of the Cell, F-69007, Lyon, France; 2Laboratoire de Biométrie et Biologie Évolutive, Université Lyon 1, UMR CNRS 5558, INRIA Erable, Villeurbanne, F-69622, France; 3Institut NeuroMyoGène, CNRS UMR 5310, INSERM U1217, Université Lyon 1, Lyon, F-69007 France Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing var- iation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named Exon Ontology, based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encod- ed protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splic- ing.
    [Show full text]
  • Integrative Genomic Analysis of Hnrnp L Splicing Regulation
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Terrae Incognitae: Integrative Genomic Analysis of Hnrnp L Splicing Regulation Brian Sebastian Cole University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Allergy and Immunology Commons, Biochemistry Commons, Bioinformatics Commons, Immunology and Infectious Disease Commons, and the Medical Immunology Commons Recommended Citation Cole, Brian Sebastian, "Terrae Incognitae: Integrative Genomic Analysis of Hnrnp L Splicing Regulation" (2015). Publicly Accessible Penn Dissertations. 1664. https://repository.upenn.edu/edissertations/1664 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1664 For more information, please contact [email protected]. Terrae Incognitae: Integrative Genomic Analysis of Hnrnp L Splicing Regulation Abstract Alternative splicing is a critical component of human gene control that generates functional diversity from a limited genome. Defects in alternative splicing are associated with disease in humans. Alternative splicing is regulated developmentally and physiologically by the combinatorial actions of cis- and trans- acting factors, including RNA binding proteins that regulate splicing through sequence-specific interactions with pre-mRNAs. In T cells, the splicing regulator hnRNP L is an essential factor that regulates alternative splicing of physiologically important mRNAs, however the broader physical and functional impact of hnRNP L remains unknown. In this dissertation, I present analysis of hnRNP L-RNA interactions with CLIP-seq, which identifies transcriptome-wide binding sites and uncovers novel functional targets. I then use functional genomics studies to define pre-mRNA processing alterations induced by hnRNP L depletion, chief among which is cassette-type alternative splicing.
    [Show full text]
  • The DEAD-Box Protein P72 Regulates ERΑ-&Sol;Oestrogen
    Oncogene (2009) 28, 4053–4064 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE The DEAD-box protein p72 regulates ERa-/oestrogen-dependent transcription and cell growth, and is associated with improved survival in ERa-positive breast cancer NC Wortham1,6, E Ahamed2,6, SM Nicol1, RS Thomas2, M Periyasamy2, J Jiang2,3, AM Ochocka2, S Shousha3, L Huson4, SE Bray5, RC Coombes2, S Ali2 and FV Fuller-Pace1 1Centre for Oncology and Molecular Medicine, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK; 2Department of Oncology, Imperial College London, Hammersmith Hospital Campus, London, UK; 3Department of Histopathology, Imperial College London, Charing Cross Hospital, London, UK; 4Statistical Advisory Service, Imperial College London, South Kensington Campus, London, UK and 5Tayside Tissue Bank, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK The DEAD-box RNA helicases p68 (DDX5) and p72 Introduction (DDX17) have been shown to act as transcriptional co- activators for a diverse range of transcription factors, The DEAD-box subfamily of RNA helicases, originally including oestrogen receptor-a (ERa). Here, we show that, so named on the basis of the presence of a conserved although both proteins interact with and co-activate ERa motif having the sequence Asp-Glu-Ala-Asp, have been in reporter gene assays, small interfering RNA-mediated implicated in cellular processes involving the regulation knockdown of p72, but not p68, results in a significant of RNA structure, including pre-mRNA processing, inhibition of oestrogen-dependent transcription of endo- RNA export, RNA degradation, ribosome assembly, genous ERa-responsive genes and oestrogen-dependent translation and miRNA maturation (Linder et al., 1989; growth of MCF-7 and ZR75-1 breast cancer cells.
    [Show full text]
  • Regulation of Mirna Biogenesis and Histone Modification by K63-Polyubiquitinated
    Author Manuscript Published OnlineFirst on March 15, 2019; DOI: 10.1158/0008-5472.CAN-18-2376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Regulation of miRNA biogenesis and histone modification by K63-polyubiquitinated DDX17 controls cancer stem-like features Shih-Han Kao1,2, Wei-Chung Cheng1,2,3, Yi-Ting Wang5, Han-Tsang Wu6, Han-Yu Yeh1, Yu-Ju Chen5, Ming-Hsui Tsai7, Kou-Juey Wu1,2,3,4,8,9,10* 1Research Center for Tumor Medical Science, China Medical University, Taichung 404, Taiwan; 2Drug Development Center, China Medical University, Taichung 404, Taiwan; 3Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404, Taiwan; 4Institute of New Drug Development, China Medical University, Taichung 404, Taiwan; 5Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan; 6Department of Cell and Tissue Engineering, Changhua Christian Hospital, Changhua City 500, Taiwan; 7Department of Otolaryngology, China Medical University Hospital, Taichung 404, Taiwan; 8Department of Medical Research, China Medical University Hospital, Taichung 404, Taiwan; 9Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan; 10Cancer Genome Research Center, Chang Gung Memorial Hospital at Linkou, Taoyuan 333, Taiwan Correspondence should be addressed to: Kou-Juey Wu, Research Center for Tumor Medical Science, Institute of New Drug Development, China Medical University, Taichung 40402, Taiwan; Email: [email protected] or 1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 15, 2019; DOI: 10.1158/0008-5472.CAN-18-2376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
    [Show full text]
  • Human Proteins That Interact with RNA/DNA Hybrids
    Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Resource Human proteins that interact with RNA/DNA hybrids Isabel X. Wang,1,2 Christopher Grunseich,3 Jennifer Fox,1,2 Joshua Burdick,1,2 Zhengwei Zhu,2,4 Niema Ravazian,1 Markus Hafner,5 and Vivian G. Cheung1,2,4 1Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA; 2Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA; 3Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland 20892, USA; 4Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109, USA; 5Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892, USA RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing. The proteins are enriched for K homology (KH) and helicase domains. Among them, more than 300 proteins preferred binding to hybrids than double-stranded DNA. These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated.
    [Show full text]
  • RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by Mirna-Based Repression of RYBP and Its PRC1-Dependent and -Independent Functions
    Article RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by miRNA-Based Repression of RYBP and its PRC1-Dependent and -Independent Functions Graphical Abstract Authors Huanhuan Li, Ping Lai, Jinping Jia, ..., Duanqing Pei, Andrew P. Hutchins, Hongjie Yao Correspondence [email protected] In Brief RNA-binding proteins have poorly defined roles in somatic cell reprogramming. Li et al. show that the RNA-binding protein DDX5 erects an epigenetic barrier to reprogramming. DDX5 controls RYBP through microRNA- 125b to suppress specific somatic genes through deposition of H2AK119ub1 while activating an OCT4-KDM2B pluripotent gene program. Highlights d DDX5 acts as a barrier to somatic cell reprogramming d DDX5 loss of function upregulates RYBP through microRNA-125b d Upregulated RYBP enhances H2AK119ub1 deposition at lineage-specific genes via PRC1 d DDX5 silencing activates the OCT4-KDM2B network through RYBP independently of PRC1 Li et al., 2017, Cell Stem Cell 20, 462–477 April 6, 2017 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2016.12.002 Cell Stem Cell Article RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by miRNA-Based Repression of RYBP and its PRC1-Dependent and -Independent Functions Huanhuan Li,1,2,7 Ping Lai,1,7 Jinping Jia,3,7 Yawei Song,1 Qing Xia,1 Kaimeng Huang,1 Na He,4 Wangfang Ping,1 Jiayu Chen,5 Zhongzhou Yang,1 Jiao Li,1 Mingze Yao,1 Xiaotao Dong,1 Jicheng Zhao,6 Chunhui Hou,4 Miguel A. Esteban,1 Shaorong Gao,5 Duanqing Pei,1 Andrew P. Hutchins,4 and Hongjie Yao1,8,* 1CAS Key Laboratory of Regenerative
    [Show full text]
  • DDX5 Antibody Purified Mouse Monoclonal Antibody Catalog # Ao2243a
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 DDX5 Antibody Purified Mouse Monoclonal Antibody Catalog # AO2243a Specification DDX5 Antibody - Product Information Application E, WB, FC, IHC Primary Accession P17844 Reactivity Human, Mouse, Monkey Host Mouse Clonality Monoclonal Isotype IgG2a Calculated MW 69.1kDa KDa Description DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), are putative RNA helicases. They are implicated in a number of cellular processes involving alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. This gene encodes a DEAD box protein, which is a RNA-dependent ATPase, and also a proliferation-associated nuclear antigen, specifically reacting with the simian virus 40 tumor antigen. This gene consists of 13 exons, and alternatively spliced transcripts containing several intron sequences have been detected, but no isoforms encoded by these transcripts have been identified. Immunogen Purified recombinant fragment of human DDX5 (AA: 475-614) expressed in E. Coli. Formulation Purified antibody in PBS with 0.05% sodium azide DDX5 Antibody - Additional Information Gene ID 1655 Page 1/3 10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 Other Names Probable ATP-dependent RNA helicase DDX5, 3.6.4.13, DEAD box protein 5, RNA helicase p68, DDX5, G17P1, HELR, HLR1 Dilution E~~1/10000 WB~~1/500 - 1/2000 FC~~1/200 - 1/400 IHC~~1/200 - 1/1000 Storage Maintain refrigerated at 2-8°C for up to 6 months.
    [Show full text]
  • Ddx17 RNA Helicases in the Control of the Pro-Migratory NFAT5 Transcription Factor
    Oncogene (2012) 31, 4536–4549 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Dual role of the ddx5/ddx17 RNA helicases in the control of the pro-migratory NFAT5 transcription factor S Germann1,2,3,4,5, L Gratadou1,2,3,4,5, E Zonta1,2,3,4,5, E Dardenne1,2,3,4,5, B Gaudineau6, M Fouge` re6, S Samaan1,2,3,4,5, M Dutertre1,2,3,4,5, S Jauliac6 and D Auboeuf1,2,3,4,5 1Universite´ de Lyon, Lyon, France; 2Inserm U1052, Lyon, France; 3CNRS UMR5286, Lyon, France; 4Centre de Recherche en Cance´rologie de Lyon, Lyon, France; 5Universite´ Lyon 1, Lyon, France and 6CNRS UMR7212, INSERM U944, Universite´ Paris Diderot, Institut d’He´matologie, Hoˆpital Saint-Louis, Paris, France Ddx5 and ddx17 are two highly related RNA helicases directly linked to the migratory and invasive phenotype involved in both transcription and splicing. These of cancer cells (Friedl and Wolf, 2003). Invasion is a proteins coactivate transcription factors involved in complex process relying on the capacity of the cells to cancer such as the estrogen receptor alpha, p53 and migrate and to destroy and reorganize the extracellular beta-catenin. Ddx5 and ddx17 are part of the splicing matrix. It is now well established that tumor progression machinery and can modulate alternative splicing, the relies on the alteration of transcriptional programs main mechanism increasing the proteome diversity. controlling specific cellular programs (Hanahan and Alternative splicing also has a role in gene expression Weinberg, 2000). In this context, the NFAT family of level regulation when it is coupled to the nonsense- transcription factors is gaining increasing interest in mediated mRNA decay (NMD) pathway.
    [Show full text]
  • Global View of Candidate Therapeutic Target Genes in Hormone-Responsive Breast Cancer
    International Journal of Molecular Sciences Review Global View of Candidate Therapeutic Target Genes in Hormone-Responsive Breast Cancer 1, 1, 1 1 Annamaria Salvati y , Valerio Gigantino y, Giovanni Nassa , Valeria Mirici Cappa , Giovanna Maria Ventola 2, Daniela Georgia Cristina Cracas 2, Raffaella Mastrocinque 2, Francesca Rizzo 1 , Roberta Tarallo 1 , Alessandro Weisz 1,3,* and Giorgio Giurato 1,* 1 Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry ‘Scuola Medica Salernitana’, University of Salerno, 84081 Baronissi (SA), Italy; [email protected] (A.S.); [email protected] (V.G.); [email protected] (G.N.); [email protected] (V.M.C.); [email protected] (F.R.); [email protected] (R.T.) 2 Genomix4Life, 84081 Baronissi (SA), Italy; [email protected] (G.M.V.); [email protected] (D.G.C.C.); [email protected] (R.M.) 3 CRGS—Genome Research Center for Health, University of Salerno Campus of Medicine, 84081 Baronissi (SA), Italy * Correspondence: [email protected] (A.W.); [email protected] (G.G.); Tel.: +39-089-965043 (A.W.); +39-089-968286 (G.G.) These authors contributed equally to this work. y Received: 14 May 2020; Accepted: 3 June 2020; Published: 6 June 2020 Abstract: Breast cancer (BC) is a heterogeneous disease characterized by different biopathological features, differential response to therapy and substantial variability in long-term-survival. BC heterogeneity recapitulates genetic and epigenetic alterations affecting transformed cell behavior. The estrogen receptor alpha positive (ERα+) is the most common BC subtype, generally associated with a better prognosis and improved long-term survival, when compared to ERα-tumors.
    [Show full text]
  • Functions of DEAD Box RNA Helicases DDX5 and DDX17 in Chromatin Organization and Transcriptional Regulation
    BMB Rep. 2018; 51(12): 613-622 BMB www.bmbreports.org Reports Invited Mini Review Functions of DEAD box RNA helicases DDX5 and DDX17 in chromatin organization and transcriptional regulation Guillaume Giraud#, Sophie Terrone# & Cyril F. Bourgeois Laboratoire de Biologie et Modelisation de la Cellule, Universite de Lyon, CNRS UMR 5239, INSERM U1210, Ecole Normale Superieure de Lyon, Universite Claude Bernard Lyon 1, F-69007 Lyon, France RNA helicases DDX5 and DDX17 are multitasking proteins highly homologous ATPase domain (90% homology in human that regulate gene expression in different biological contexts proteins), and ATP binding and hydrolysis are necessary to through diverse activities. Special attention has long been paid catalyze the unwinding of local RNA secondary structures, as to their function as coregulators of transcription factors, well as for their RNA annealing activity (2-4). providing insight about their functional association with a DDX5 and DDX17 are also important coregulators of number of chromatin modifiers and remodelers. However, to various transcription factors, and they interact with a number date, the variety of described mechanisms has made it difficult of chromatin-associated factors (5). This may seem to conflict to understand precisely how these proteins work at the with their enzymatic activity, and it remains uncertain whether molecular level, and the contribution of their ATPase domain their helicase activity is required for this particular function. In to these mechanisms remains unclear as well. In light of their animals, it is not known whether these proteins have an association with long noncoding RNAs that are key epigenetic affinity for double-stranded DNA or RNA-DNA hybrids, which regulators, an emerging view is that DDX5 and DDX17 may could be relevant for chromatin and transcription-related act through modulating the activity of various ribonucleo- functions.
    [Show full text]