Detection of double-stranded RNA-protein interactions by methylene blue-mediated photo-crosslinking

RNA. 1996 Jun;2(6):611-21.

Abstract

Double-stranded(ds) RNA-binding proteins have diverse functions in the cell. An obstacle to investigating the interactions between these proteins and dsRNA is the relative inefficiency of traditional UV-crosslinking methods for extended regions of dsRNA. We have therefore developed an alternative procedure for RNA-protein photo-crosslinking that efficiently induces RNA-protein crosslinks in double-stranded regions of RNA. We show that dsRNA-protein crosslinks can be induced by visible light in the presence of the dye methylene blue, which most likely mediates crosslinking by intercalating in the dsRNA helix. A recombinant dsRNA binding domain from the Drosophila staufen protein and human protein kinase R were crosslinked by UV or methylene blue to a series of dsRNAs. In each case, the degree of crosslinking was greater with methylene blue, particularly with RNAs with few single-stranded loops. Methylene blue-mediated crosslinking therefore complements and extends the existing repertoire of crosslinking methods for detecting RNA-protein interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cross-Linking Reagents
  • Drosophila
  • Drosophila Proteins*
  • HeLa Cells
  • Humans
  • Intercalating Agents / metabolism
  • Intercalating Agents / pharmacology
  • Methylene Blue / pharmacology*
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism
  • Nucleic Acid Conformation
  • Photochemistry
  • Protein Kinases / chemistry
  • Protein Kinases / metabolism
  • RNA, Double-Stranded / chemistry
  • RNA, Double-Stranded / metabolism*
  • RNA, Viral / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Ultraviolet Rays

Substances

  • Cross-Linking Reagents
  • Drosophila Proteins
  • Intercalating Agents
  • Nuclear Proteins
  • RNA, Double-Stranded
  • RNA, Viral
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • adenovirus associated RNA
  • stau protein, Drosophila
  • Protein Kinases
  • Methylene Blue