Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22.
EBER 1, a small noncoding viral RNA abundantly expressed in all cells transformed by Epstein-Barr virus (EBV), has been shown to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of a mixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1 molecule. EMSAs performed with EBER 1 deletion constructs and EBER 1 stem-loops inserted into a nonbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1 inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1 in EBV-infected human B cells.
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Victor
V
Fok
Rachel M
RM
Mitton-Fry
Angie
A
Grech
Joan A
JA
Steitz
D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D058619:Maltose-Binding Proteins; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D022661:RNA, Untranslated; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; C099537:Epstein-Barr virus encoded RNA 1; D058619:Maltose-Binding Proteins; D022661:RNA, Untranslated; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C067169:RPL22 protein, human