Detection of double-stranded RNA-protein interactions by methylene blue-mediated photo-crosslinking.
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.
Liu
Z R
ZR
Department of Biochemistry, University of Cambridge, United Kingdom.
Wilkie
A M
AM
Clemens
M J
MJ
Smith
C W
CW
D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004330:Drosophila; D029721:Drosophila Proteins; D006367:HeLa Cells; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism / Q000494:pharmacology; D008751:Methylene Blue / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; D007364:Intercalating Agents; D009687:Nuclear Proteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C052835:adenovirus associated RNA; C069927:stau protein, Drosophila; D011494:Protein Kinases; D008751:Methylene Blue