Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22 (original) (raw)
- Victor Fok1,2,4,
- Rachel M. Mitton-Fry1,4,
- Angie Grech3, and
- Joan A. Steitz1,2
- 1Department of Molecular Biophysics and Biochemistry
- 2Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA
- 3McKinsey and Company, Inc., New York, New York 10022, USA
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.
- Epstein-Barr virus
- EBER 1
- ribosomal protein L22
- electrophoretic mobility-shift assay
- in vivo UV cross-linking
- RNA–protein interaction
Footnotes
4
↵4 These authors contributed equally to this work.Reprint requests to: Joan A. Steitz, Department of Molecular Biophysics and Biochemistry and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA; e-mail: joan.steitz{at}yale.edu; fax: (203) 624-8213.
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2339606.
- Received December 20, 2005.
- Accepted January 25, 2006.
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