Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22

RNA. 2006 May;12(5):872-82. doi: 10.1261/rna.2339606. Epub 2006 Mar 23.

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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Carrier Proteins / metabolism
  • Cell Line
  • Cross-Linking Reagents / pharmacology
  • Electrophoretic Mobility Shift Assay
  • Herpesvirus 4, Human / genetics
  • Humans
  • In Vitro Techniques
  • Maltose-Binding Proteins
  • Nucleic Acid Conformation
  • Plasmids / genetics
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Untranslated*
  • RNA, Viral / chemistry
  • RNA, Viral / genetics
  • RNA, Viral / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / isolation & purification
  • RNA-Binding Proteins / metabolism*
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / isolation & purification
  • Ribosomal Proteins / metabolism*
  • Sequence Deletion
  • Transcription, Genetic
  • Transfection
  • Ultraviolet Rays

Substances

  • Carrier Proteins
  • Cross-Linking Reagents
  • Epstein-Barr virus encoded RNA 1
  • Maltose-Binding Proteins
  • RNA, Untranslated
  • RNA, Viral
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Ribosomal Proteins
  • RPL22 protein, human