DOCSLIB.ORG

RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by Mirna-Based Repression of RYBP and Its PRC1-Dependent and -Independent Functions

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read 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 Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, CAS Center for Excellence in Molecular Cell Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China 2GZMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou 511436, China 3Laboratory of Translational Genomics, National Cancer Institute, NIH, Bethesda, MD 20892, USA 4Department of Biology, Southern University of Science and Technology of China, Shenzhen 518055, China 5School of Life Sciences and Technology, Tongji University, Shanghai 200092, China 6National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 7Co-first author 8Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2016.12.002 SUMMARY followed by the upregulation of pluripotency genes (Brambrink et al., 2008; Li et al., 2010; Samavarchi-Tehrani et al., 2010; RNA-binding proteins (RBPs), in addition to their Stadtfeld et al., 2008). The phased and timed nature of these functions in cellular homeostasis, play important transitions suggests tight regulation of the reprogramming pro- roles in lineage specification and maintaining cellular cess (Golipour et al., 2012; Polo et al., 2012). It is now clear identity. Despite their diverse and essential functions, that the OSKM factors need to overcome a series of epigenetic which touch on nearly all aspects of RNA metabolism, barriers and pass through a sequence of distinct molecular the roles of RBPs in somatic cell reprogramming are and cellular events. Tremendous effort has been focused on studying the roles of chromatin-binding proteins and modifying poorly understood. Here we show that the DEAD- enzymes in reprogramming and pluripotency (Apostolou and box RBP DDX5 inhibits reprogramming by repressing Hochedlinger, 2013; Rais et al., 2013). However, much less has the expression and function of the non-canonical pol- been reported regarding the roles of RNA-binding proteins ycomb complex 1 (PRC1) subunit RYBP. Disrupting (RBPs) in reprogramming, pluripotency, and differentiation Ddx5 expression improves the efficiency of iPSC gen- despite the ability of the RBP LIN28A, to replace KLF4 in human eration and impedes processing of miR-125b, leading reprogramming (Yu et al., 2007), suggesting that other RBPs to Rybp upregulation and suppression of lineage- may also have critical roles in regulating the reprogramming specific genes via RYBP-dependent ubiquitination process. of H2AK119. Furthermore, RYBP is required for RBPs are involved in a wide range of regulatory pathways, PRC1-independent recruitment of OCT4 to the pro- from RNA metabolism to epigenetic regulation (Guallar and moter of Kdm2b, a histone demethylase gene that Wang, 2014). Importantly, an increasing number of studies have demonstrated that RBPs not only play constitutive roles promotes reprogramming by reactivating endoge- but also have important functions in the maintenance of cell iden- nous pluripotency genes. Together, these results tity (Zhang et al., 2016). DEAD-box RBPs have essential roles in reveal important functions of DDX5 in regulating re- cellular RNA metabolism, including transcription, pre-mRNA programming and highlight the importance of a splicing, ribosome biogenesis, transport, translation, and RNA Ddx5-miR125b-Rybp axis in controlling cell fate. decay (Linder and Jankowsky, 2011). DEAD-box RBP DDX5 is a component of the Drosha complex and plays important roles in regulating microRNA processing INTRODUCTION (Dardenne et al., 2014; Gregory et al., 2004). DDX5 associates with the long non-coding RNA (lncRNA) RNA component of Induced pluripotent stem cells (iPSCs) provide a powerful in vitro mitochondrial RNAase P (RMRP) and modulates Th17 cell experimental model to investigate the mechanisms controlling effector functions (Huang et al., 2015) and regulates CCCTC- cell fate conversion. The transduction of the ‘‘Yamanaka factors’’ binding factor (CTCF) transcriptional insulation (Yao et al., OCT4, SOX2, KLF4, and c-MYC (OSKM) into somatic cells 2010). In addition, p53 and SMAD proteins can both modulate sets off a series of phased transitions, initiated by the reduction microRNA metabolism through their association with DDX5 of somatic genes, a mesenchymal-epithelial transition (MET), (Davis et al., 2008; Suzuki et al., 2009). 462 Cell Stem Cell 20, 462–477, April 6, 2017 ª 2016 Elsevier Inc. AB C OSKM Ddx5 mRNA Ddx5 mRNA *** *** 1.2 shDdx5 #1 shDdx5 #2 10 *** shCtrl *** 1 level 8 0.8 AP staining 6 0.6 4 0.4 2 0.2 Oct4-GFP Oct4-GFP 12.4% Oct4-GFP Relative expression level 4.10% 13.7% Relative expression 0 0 FACS MEFs iPSCs shCtrl mESCs shDdx5 shDdx5 #1 #2 α-DDX5 α-DDX5 FSC GFP α-β-Actin α-β-Actin D E F OSKM OSKM Secondary MEFs +Dox +Vc Ctrl DDX5 *** 350 *** *** 600 *** 300 AP staining 250 400 200 150 5.45% 1.02% Oct4-GFP Oct4-GFP 100 200 FACS 50 Oct4-GFP colony number Oct4-GFP colony number 0 0 FSC #1 #2 #1 #2 shCtrl shCtrl GFP Ddx5 Ddx5 Ddx5 Ddx5 h sh sh sh s G OSKM HISecondary MEFs 100 OSKM +Dox +Vc ** OSKM+DDX5 ** 100 *** 80 180 80 ** 60 135 60 40 90 40 20 Oct4-GFP colony number *** 45 20 0 Oct4-GFP colony number Oct4-GFP colony number D0 D3 D6 D9 D12 0 0 Ctrl DDX5 rl 5 t X C D D J K MEFs MEFs OSKM+shCtrl 1000 OSKM 10000 OSKM+DDX5 OSKM+shDdx5 #1 *** *** *** 1000 OSKM+shDdx5 #2 *** level 100 100 *** *** ression level 10 xp Snai1 Zeb2 10 1 ** *** ee v Epcam Krt8 Snai1 Zeb2 ti 0.1 a Epithelial genes 1 Rel 0.01 Relative expression Epcam Cldn3 0.001 0.1 Mesenchymal genes Epithelial genes 0.0001 Mesenchymal genes Figure 1. DDX5 Loss of Function Promotes Somatic Cell Reprogramming (A) qRT-PCR and western blot analysis for endogenous Ddx5 mRNA and protein levels in MEFs, mESCs, and iPSCs. (B) qRT-PCR and western blot to test DDX5 knockdown efficiency in MEFs transduced with OSKM together with either control shRNA or two Ddx5 shRNAs. (C) Top: AP-stained wells of a representative reprogramming experiment transfected with OSKM and either a control or Ddx5-targeting shRNA on day 9. Bottom: GFP+ cells analyzed by fluorescence-activated cell sorting (FACS) in reprogramming cells transfected with OSKM and either a control or Ddx5-targeting shRNA on day 12. (legend continued on next page) Cell Stem Cell 20, 462–477, April 6, 2017 463 In this study, we showed that DDX5 is a barrier for somatic cell Next, we transfected DDX5 along with OSKM into MEFs and reprogramming. DDX5 loss of function upregulates the non-ca- assessed the effects of DDX5 overexpression in MEFs (Fig- nonical PRC1 component RING1 and YY1 binding protein ure S1B). DDX5 significantly inhibited the formation of both (RYBP) through a mechanism involving microRNA (miRNA) AP+ and GFP+ colonies compared with the controls (Figures 125b, which leads to increased RYBP-mediated ubiquitination 1F and 1G). Furthermore, we observed that DDX5 overexpres- of histone H2A at K119 (H2AK119ub1) at lineage-specific loci sion significantly inhibited the timing of the appearance of iPSCs and repression of these genes. DDX5 loss of function enhances (Figure 1H). In addition, we observed a similar result with DDX5 reprogramming efficiency and facilitates RYBP-mediated OCT4 overexpression in the secondary MEF system (Figure 1I). The recruitment at the Kdm2b locus, which promotes reprogram- blocking effect of DDX5 during reprogramming was verified ming. This effect is independent of the RYBP-PRC1 complex with another overexpression system (pW-TRE tet-on inducible and, instead, an OCT4-RYBP complex forms, illustrating system) (Figures S1C–S1E). Consistent with the knockdown ex- the context-specific functions of RYBP. DDX5 loss of func- periments, exogenous DDX5 did not affect cell proliferation in the tion not only results in the enrichment of RYBP-stimulated presence of OSKM induction, indicating a growth-independent H2AK119ub1 at lineage-specific genes, thus suppressing effect of DDX5 on reprogramming efficiency (Figures S1F and the expression of these genes, but also activates the OCT4- S1G). In the early stage of reprogramming, the cells pass KDM2B pluripotent network and so assists reprogramming through a MET (Li et al., 2010; Samavarchi-Tehrani et al., cells to overcome an epigenetic barrier in the late phase of 2010).
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]
  • 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]
  • Intragenic Recruitment of NF-κB Drives Splicing Modifications Upon
    ARTICLE https://doi.org/10.1038/s41467-020-16853-x OPEN Intragenic recruitment of NF-κB drives splicing modifications upon activation by the oncogene Tax of HTLV-1 Lamya Ben Ameur 1, Paul Marie 1,8, Morgan Thenoz1,2,8, Guillaume Giraud1,8, Emmanuel Combe 1, Jean-Baptiste Claude1, Sebastien Lemaire1, Nicolas Fontrodona1, Hélène Polveche3, Marine Bastien1,4, ✉ ✉ Antoine Gessain5, Eric Wattel 6,7, Cyril F. Bourgeois 1, Didier Auboeuf 1 & Franck Mortreux 1 1234567890():,; Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB–responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases. 1 Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, 46 Allée d’Italie Site Jacques Monod, 69007 Lyon, France.
    [Show full text]