title: AUF1/hnRNP D is a novel protein partner of the EBER1 noncoding RNA of Epstein-Barr virus.
abstract: Epstein-Barr virus (EBV)-infected cells express two noncoding RNAs called EBV-encoded RNA (EBER) 1 and EBER2. Despite their high abundance in the nucleus (about 10(6) copies), the molecular function of these noncoding RNAs has remained elusive. Here, we report that the insertion into EBER1 of an RNA aptamer that binds the bacteriophage MS2 coat protein allows the isolation of EBER1 and associated protein partners. By combining MS2-mediated selection with stable isotope labeling of amino acids in cell culture (SILAC) and analysis by mass spectrometry, we identified AUF1 (AU-rich element binding factor 1)/hnRNP D (heterogeneous nuclear ribonucleoprotein D) as an interacting protein of EBER1. AUF1 exists as four isoforms generated by alternative splicing and is best known for its role in destabilizing mRNAs upon binding to AU-rich elements (AREs) in their 3' untranslated region (UTR). Using UV crosslinking, we demonstrate that predominantly the p40 isoform of AUF1 interacts with EBER1 in vivo. Electrophoretic mobility shift assays show that EBER1 can compete for the binding of the AUF1 p40 isoform to ARE-containing RNA. Given the high abundance of EBER1 in EBV-positive cells, EBER1 may disturb the normal homeostasis between AUF1 and ARE-containing mRNAs or compete with other AUF1-interacting targets in cells latently infected by EBV.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: Nara
authors.initials: N
authors.lastname: Lee
authors.forename: Genaro
authors.initials: G
authors.lastname: Pimienta
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D020413:3' Untranslated Regions; D063307:AU Rich Elements; D052157:Aptamers, Nucleotide / Q000235:genetics; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000737:chemistry / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D047468:Immunoprecipitation; D016254:Mutagenesis, Insertional; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D020871:RNA Stability; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D052157:Aptamers, Nucleotide; C099537:Epstein-Barr virus encoded RNA 1; C000630986:HNRNPD protein, human; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D020033:Protein Isoforms; D012367:RNA, Viral