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

Abstract

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.

Expression of a Novel Leishmania Gene Encoding a Histone H1-Like Protein in Leishmania major Modulates Parasite Infectivity In Vitro

“…In order to demonstrate that LNP18, which has characteristics of a DNA-binding protein, does interact with Leishmania DNA, we used the UV-cross-linking hybridization technique (41). Nuclear extracts were irradiated as described in Materials and Methods.…”

“…For in vitro cross-linking of DNA to proteins, we used the approach used by Pelle and Murphy (41). Briefly, isolated nuclei from approximately 5 ϫ 10 9 L. major promastigotes were homogenized on ice in 200 l of homogenization buffer (25 mM Tris-HCl, 1 mM EDTA; pH 8) in a Dounce homogenizer.…”

Potential role for snoRNAs in PKR activation during metabolic stress

“…UV-C irradiation is a "zero length" cross-linking technique that will only covalently link RNA to proteins that are in direct contact and, further, will not create protein-protein cross-links (23,24). Thus, these results indicate that PKR is directly interacting with snoRNAs in cells.…”

Mapping of RNA–protein interactions

“…Such linkages can generally be achieved by UV or chemical means. The complexes obtained by these linkages are resistant to heat, detergent and alkali, also faster, minimizes the nonspecific interactions [24,25]. Contact-site cross-linking methods were shown to crosslink macromolecules such as protein and RNA [26,27].…”

“…Among chemical and UV cross-linking strategies, cross-linking by lengthier chemical reagent may disrupt RNA and protein complexes. UV cross-linking may not disrupt these complexes [25,35]. Generally, cross-linkages have proven to have very specific contact, yielding residue-residue contact information and increasing the cohesive strength.…”