The Mammalian Ecdysoneless Protein Interacts with RNA Helicase DDX39A To Regulate Nuclear mRNA Export

The mammalian orthologue of ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of endoplasmic reticulum stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. ABSTRACT The mammalian orthologue of ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of endoplasmic reticulum stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. We find that ECD is involved in RNA export through its interaction with DDX39A. ECD knockdown (KD) blocks mRNA export from the nucleus to the cytoplasm, which is rescued by expression of full-length ECD but not an ECD mutant that is defective in interaction with DDX39A. We have previously shown that ECD protein is overexpressed in ErbB2+ breast cancers (BC). In this study, we extended the analyses to two publicly available BC mRNA The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data sets. In both data sets, ECD mRNA overexpression correlated with short patient survival, specifically ErbB2+ BC. In the METABRIC data set, ECD overexpression also correlated with poor patient survival in triple-negative breast cancer (TNBC). Furthermore, ECD KD in ErbB2+ BC cells led to a decrease in ErbB2 mRNA level due to a block in its nuclear export and was associated with impairment of oncogenic traits. These findings provide novel mechanistic insight into the physiological and pathological functions of ECD.

[1]  S. Seno,et al.  URH49 exports mRNA by remodeling complex formation and mediating the NXF1-dependent pathway. , 2020, Biochimica et biophysica acta. Gene regulatory mechanisms.

[2]  S. Stagg,et al.  Yeast R2TP Interacts with Extended Termini of Client Protein Nop58p , 2019, Scientific Reports.

[3]  I. Ellis,et al.  A key genomic subtype associated with lymphovascular invasion in invasive breast cancer , 2019, British Journal of Cancer.

[4]  L. Pearl,et al.  Advances on the Structure of the R2TP/Prefoldin-like Complex. , 2018, Advances in experimental medicine and biology.

[5]  T. Jensen,et al.  A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA , 2018, Cell reports.

[6]  Tong Zhang,et al.  DDX39 promotes hepatocellular carcinoma growth and metastasis through activating Wnt/β-catenin pathway , 2018, Cell Death & Disease.

[7]  Wenjun Chang,et al.  [Relationship between the expression of DDX39 protein and prognosis of colorectal cancer]. , 2018, Zhonghua wei chang wai ke za zhi = Chinese journal of gastrointestinal surgery.

[8]  Michaela Müller-McNicoll,et al.  Nuclear retention of mRNAs - quality control, gene regulation and human disease. , 2017, Seminars in cell & developmental biology.

[9]  V. Wickramasinghe,et al.  Nuclear export of RNA: Different sizes, shapes and functions. , 2017, Seminars in cell & developmental biology.

[10]  F. Urano,et al.  Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response , 2017, Molecular and Cellular Biology.

[11]  Z. Hořejší,et al.  Assembly of the U5 snRNP component PRPF8 is controlled by the HSP90/R2TP chaperones , 2017, The Journal of cell biology.

[12]  K. Borden,et al.  A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery , 2017, RNA.

[13]  B. Coulombe,et al.  R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein , 2017, Nature Communications.

[14]  Hisashi Tanaka,et al.  Palindromic amplification of the ERBB2 oncogene in primary HER2-positive breast tumors , 2017, Scientific Reports.

[15]  S. Aparicio,et al.  The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation. , 2017, Biochemical and biophysical research communications.

[16]  H. Gautrey,et al.  How is Herstatin, a tumor suppressor splice variant of the oncogene HER2, regulated? , 2017, RNA biology.

[17]  De Chen,et al.  Amplification of ACK1 promotes gastric tumorigenesis via ECD-dependent p53 ubiquitination degradation , 2015, Oncotarget.

[18]  V. Band,et al.  Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation , 2016, Molecular and Cellular Biology.

[19]  Nicholas P. Schafer,et al.  Biophysical characterization and modeling of human Ecdysoneless (ECD) protein supports a scaffolding function , 2016, AIMS biophysics.

[20]  M. Kusunoki,et al.  Identification of DDX39A as a Potential Biomarker for Unfavorable Neuroblastoma Using a Proteomic Approach , 2016, Pediatric blood & cancer.

[21]  R. A. Mir,et al.  A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression , 2015, Molecular and Cellular Biology.

[22]  E. Bertrand,et al.  SnoRNPs, ZNHIT proteins and the R2TP pathway , 2015, Oncotarget.

[23]  Guangchuang Yu,et al.  ACK1 promotes gastric cancer epithelial–mesenchymal transition and metastasis through AKT–POU2F1–ECD signalling , 2015, The Journal of pathology.

[24]  V. Band,et al.  Cell type of origin as well as genetic alterations contribute to breast cancer phenotypes , 2015, Oncotarget.

[25]  Simon M Lin,et al.  The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells , 2015, Cell cycle.

[26]  S. H. Stubbs,et al.  Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy , 2014, Nucleic acids research.

[27]  Y. Kakihara,et al.  The R2TP chaperone complex: its involvement in snoRNP assembly and tumorigenesis , 2014, Biomolecular concepts.

[28]  A. Hamburger,et al.  Heregulin Negatively Regulates Transcription of ErbB2/3 Receptors via an AKT‐Mediated Pathway , 2014, Journal of cellular physiology.

[29]  D. Govender,et al.  Elevated expression of the nuclear export protein, Crm1 (exportin 1), associates with human oesophageal squamous cell carcinoma. , 2014, Oncology reports.

[30]  T. Lamkemeyer,et al.  Unexpected Role of the Steroid-Deficiency Protein Ecdysoneless in Pre-mRNA Splicing , 2014, PLoS genetics.

[31]  S. Smerdon,et al.  Phosphorylation-Dependent PIH1D1 Interactions Define Substrate Specificity of the R2TP Cochaperone Complex , 2014, Cell reports.

[32]  C. Langford,et al.  Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP , 2014, Nucleic acids research.

[33]  N. Aceto,et al.  Mammary tumor formation and metastasis evoked by a HER2 splice variant. , 2013, Cancer research.

[34]  K. Borden,et al.  Aiding and abetting cancer: mRNA export and the nuclear pore. , 2013, Trends in cell biology.

[35]  Judy M. Anderson,et al.  Overexpression of Ecdysoneless in Pancreatic Cancer and Its Role in Oncogenesis by Regulating Glycolysis , 2012, Clinical Cancer Research.

[36]  H. Wanibuchi,et al.  DDX39 acts as a suppressor of invasion for bladder cancer , 2012, Cancer science.

[37]  F. Markowetz,et al.  The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups , 2012, Nature.

[38]  A. Mayeda,et al.  Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles. , 2012, RNA.

[39]  H. Tsuda,et al.  Clinical proteomics identified ATP-dependent RNA helicase DDX39 as a novel biomarker to predict poor prognosis of patients with gastrointestinal stromal tumor. , 2012, Journal of proteomics.

[40]  S. H. Stubbs,et al.  Viral Factors Reveal a Role for REF/Aly in Nuclear RNA Stability , 2012, Molecular and Cellular Biology.

[41]  W. Houry,et al.  The R2TP complex: discovery and functions. , 2012, Biochimica et biophysica acta.

[42]  K. Borden,et al.  mRNA export and cancer , 2012, Wiley interdisciplinary reviews. RNA.

[43]  I. Ellis,et al.  Overexpression of a novel cell cycle regulator ecdysoneless in breast cancer: a marker of poor prognosis in HER2/neu-overexpressing breast cancer patients , 2012, Breast Cancer Research and Treatment.

[44]  Donald W Kufe,et al.  CBS9106 is a novel reversible oral CRM1 inhibitor with CRM1 degrading activity. , 2011, Blood.

[45]  I. Chung,et al.  Requirement of DDX39 DEAD box RNA helicase for genome integrity and telomere protection , 2011, Aging cell.

[46]  T. Stamminger,et al.  The Cellular DExD/H-Box RNA-Helicases UAP56 and URH49 Exhibit a CRM1-Independent Nucleocytoplasmic Shuttling Activity , 2011, PloS one.

[47]  Ed Hurt,et al.  Linking gene regulation to mRNA production and export. , 2011, Current opinion in cell biology.

[48]  A. Lamond,et al.  Nuclear speckles. , 2011, Cold Spring Harbor perspectives in biology.

[49]  Zhi-Ming Zheng,et al.  Requirement of UAP56, URH49, RBM15, and OTT3 in the expression of Kaposi sarcoma-associated herpesvirus ORF57. , 2010, Virology.

[50]  A. Lamond,et al.  HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. , 2010, Molecular cell.

[51]  V. Wickramasinghe,et al.  GANP enhances the efficiency of mRNA nuclear export in mammalian cells , 2010, Nucleus.

[52]  S. Masuda,et al.  The Closely Related RNA helicases, UAP56 and URH49, Preferentially Form Distinct mRNA Export Machineries and Coordinately Regulate Mitotic Progression , 2010, Molecular biology of the cell.

[53]  W. Bresnahan,et al.  Binding of the Human Cytomegalovirus (HCMV) Tegument Protein UL69 to UAP56/URH49 Is Not Required for Efficient Replication of HCMV , 2010, Journal of Virology.

[54]  V. Band,et al.  Biochemical characterization of human Ecdysoneless reveals a role in transcriptional regulation , 2010, Biological chemistry.

[55]  C. Ponting,et al.  UIF, a New mRNA Export Adaptor that Works Together with REF/ALY, Requires FACT for Recruitment to mRNA , 2009, Current Biology.

[56]  Jenny G. Parvani,et al.  An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance , 2009, Molecular Cancer Therapeutics.

[57]  V. Band,et al.  Role of Mammalian Ecdysoneless in Cell Cycle Regulation* , 2009, The Journal of Biological Chemistry.

[58]  P. Thibault,et al.  Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export‐competent RNP , 2009, The EMBO journal.

[59]  Ed Hurt,et al.  Adaptor Aly and co‐adaptor Thoc5 function in the Tap‐p15‐mediated nuclear export of HSP70 mRNA , 2009, The EMBO journal.

[60]  P. Silver,et al.  mRNA nuclear export and human disease , 2008, Disease Models & Mechanisms.

[61]  Ed Hurt,et al.  Exporting RNA from the nucleus to the cytoplasm , 2007, Nature Reviews Molecular Cell Biology.

[62]  T. Sugiura,et al.  DDX39, upregulated in lung squamous cell cancer, displays RNA helicase activities and promotes cancer cell growth , 2007, Cancer biology & therapy.

[63]  Ed Hurt,et al.  Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2. , 2007, Molecular cell.

[64]  R. Kehlenbach,et al.  CRM1-mediated nuclear export: to the pore and beyond. , 2007, Trends in cell biology.

[65]  T. Sugiura,et al.  Intracellular characterization of DDX39, a novel growth-associated RNA helicase. , 2007, Experimental cell research.

[66]  Sam W. Lee,et al.  The human orthologue of Drosophila ecdysoneless protein interacts with p53 and regulates its function. , 2006, Cancer research.

[67]  M. Campiglio,et al.  Role of exon-16-deleted HER2 in breast carcinomas. , 2006, Endocrine-related cancer.

[68]  Seiji Masuda,et al.  Recruitment of the human TREX complex to mRNA during splicing. , 2005, Genes & development.

[69]  V. Henrich,et al.  Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis , 2004, Development.

[70]  L. Johnson,et al.  Growth-regulated expression and G0-specific turnover of the mRNA that encodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56. , 2004, Nucleic acids research.

[71]  B. Cullen.  Assaying nuclear messenger RNA export in human cells. , 2004, Methods in molecular biology.

[72]  B. Cullen,et al.  Nuclear mRNA export: insights from virology. , 2003, Trends in biochemical sciences.

[73]  J. Steitz,et al.  Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock. , 2001, RNA.

[74]  Ed Hurt,et al.  A Conserved mRNA Export Machinery Coupled to pre-mRNA Splicing , 2002, Cell.

[75]  B. Cullen,et al.  Formation of Tap/NXT1 Heterodimers Activates Tap-Dependent Nuclear mRNA Export by Enhancing Recruitment to Nuclear Pore Complexes , 2002, Molecular and Cellular Biology.

[76]  M. Mann,et al.  Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly , 2001, Nature.

[77]  M. Denton,et al.  Expression of herstatin, an autoinhibitor of HER-2/neu, inhibits transactivation of HER-3 by HER-2 and blocks EGF activation of the EGF receptor , 2001, Oncogene.

[78]  D. Tollervey,et al.  Mex67p Mediates Nuclear Export of a Variety of RNA Polymerase II Transcripts* , 2000, The Journal of Biological Chemistry.

[79]  B. Cullen,et al.  The Human Tap Nuclear RNA Export Factor Contains a Novel Transportin-dependent Nuclear Localization Signal That Lacks Nuclear Export Signal Function* , 1999, The Journal of Biological Chemistry.

[80]  B. Cullen,et al.  Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear RNA export factor. , 1999, Virology.

[81]  J. Adelman,et al.  The HER-2/neu receptor tyrosine kinase gene encodes a secreted autoinhibitor. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[82]  A. Podtelejnikov,et al.  The Mex67p‐mediated nuclear mRNA export pathway is conserved from yeast to human , 1999, The EMBO journal.

[83]  B. Cullen,et al.  The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. , 1999, Genes & development.

[84]  R. Cardiff,et al.  Elevated expression of activated forms of Neu/ErbB‐2 and ErbB‐3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer , 1999, The EMBO journal.

[85]  E. Hurt,et al.  Transfer RNA biogenesis: A visa to leave the nucleus , 1999, Current Biology.

[86]  Helena Santos-Rosa,et al.  Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores , 1998, Molecular and Cellular Biology.

[87]  M. Rosbash,et al.  Nuclear RNA export. , 1998, Genes & development.

[88]  M. Hung,et al.  A novel splice variant of HER2 with increased transformation activity , 1998, Molecular carcinogenesis.

[89]  R. Lührmann,et al.  Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores , 1997, The EMBO journal.

[90]  R. Wepf,et al.  A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution. , 1994, The EMBO journal.

[91]  E. Hurt,et al.  Nup145p is required for nuclear export of mRNA and binds homopolymeric RNA in vitro via a novel conserved motif , 1994, Cell.

[92]  M. Grooteclaes,et al.  Expression of the c-erbB2 gene in the BT474 human mammary tumor cell line: measurement of c-erbB2 mRNA half-life. , 1993, Oncogene.

[93]  W Godolphin,et al.  Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. , 1989, Science.