In vivo UV-cross-linking hybridization: a powerful technique for isolating RNA binding proteins. Application to trypanosome mini-exon derived RNA.

摘要

Differential gene expression in cells achieved, in part, through direct RNA-protein interactions. Methods for the identification of RNA binding proteins require cross-linking of proteins to RNA by chemicals or ultraviolet (UV) light followed by chromatography or density-gradient centrifugation (7,11,16). We have developed a simplified method for the rapid and efficient identification of potential regulatory RNA binding proteins. In this method, irradiation of cells with UV light induces cross-links between RNA and proteins in close contact (7,11). Boiling of extracts from irradiated cells in the presence of sodium dodecyl sulfate dissociates any non-specific RNA-protein interactions (11). After analysis of the cell extracts by SDS-PAGE, followed by Western blotting onto a nitrocellulose membrane and washing of the filter, we have found that only RNA molecules that are covalently bound to proteins are retained on the filter. Hybridization of this Western blot with an appropriate nucleic acid probe allows detection of bands of RNA-protein complexes. Antisera against the binding proteins are raised by immunizing mice with a region of the nitrocellulose membrane containing the bands of RNA-protein complexes. Using this approach we have found that in African trypanosomes, mini-exon derived RNA transcripts form complexes with cytoplasmic binding proteins in different life cycle stages of the parasite. Evidence for the specificity of mini-exon derived RNA-protein interactions is shown using in vitro UV-cross-linking analysis in which only in vitro generated sense (but not antisense) mini-exon derived RNA transcripts form complexes with cytoplasmic proteins.