INVIVO UV-CROSS-LINKING HYBRIDIZATION - A POWERFUL TECHNIQUE FOR ISOLATING RNA-BINDING PROTEINS - APPLICATION TO TRYPANOSOME MINI-EXON DERIVED RNA

Differential gene expression in cells is achieved, in part, through di rect RNA-protein interactions. Methods for the identification of RNA b inding proteins require cross-linking of proteins to RNA by chemicals or ultraviolet (UV) light followed by chromatography or density-gradie nt centrifugation (7,11,16). We have developed a simplified method for the rapid and efficient identification of potential regulatory RNA bi nding proteins. In this method, irradiation of cells with UV light ind uces cross-links between RNA and proteins in close contact (7,11). Boi ling of extracts from irradiated cells in the presence of sodium dodec yl 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 protei ns are retained on the filter. Hybridization of this Western blot with an appropriate nucleic acid probe allows detection of bands of RNA-pr otein complexes. Antisera against the binding proteins are raised by i mmunizing 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 st ages of the parasite. Evidence for the specificity of mini-exon derive d RNA-protein interactions is shown using in vitro UV-cross-linking an alysis in which only in vitro generated sense (but not antisense) mini -exon derived RNA transcripts form complexes with cytoplasmic proteins .