Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Binding Sites
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Carrier Proteins / metabolism
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Cell Line
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Cross-Linking Reagents / pharmacology
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Electrophoretic Mobility Shift Assay
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Herpesvirus 4, Human / genetics
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Humans
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In Vitro Techniques
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Maltose-Binding Proteins
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Nucleic Acid Conformation
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Plasmids / genetics
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Protein Binding
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Protein Structure, Tertiary
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RNA, Untranslated*
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RNA, Viral / chemistry
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RNA, Viral / genetics
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RNA, Viral / metabolism*
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / isolation & purification
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RNA-Binding Proteins / metabolism*
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Ribosomal Proteins / genetics
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Ribosomal Proteins / isolation & purification
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Ribosomal Proteins / metabolism*
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Sequence Deletion
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Transcription, Genetic
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Transfection
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Ultraviolet Rays
Substances
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Carrier Proteins
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Cross-Linking Reagents
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Epstein-Barr virus encoded RNA 1
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Maltose-Binding Proteins
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RNA, Untranslated
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RNA, Viral
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RNA-Binding Proteins
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Recombinant Fusion Proteins
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Ribosomal Proteins
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RPL22 protein, human