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

• Published in: RNA (Cambridge) Vol. 2; no. 6; pp. 611 - 621
• Main Authors: Liu, Z R, Wilkie, A M, Clemens, M J, Smith, C W
• Format: Journal Article
• Language: English
• Published: United States 01.06.1996
• Subjects: 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; Recombinant Fusion Proteins - metabolism; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - metabolism; RNA, Viral - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Ultraviolet Rays
• ISSN: 1355-8382; 1469-9001

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.