Analysis of site-specific protein–RNA cross-links in isolated RNP complexes, combining affinity selection and mass spectrometry

An important aspect of the assembly of RNPs, and in particular of spliceosomes, is the succession of proteins bound to any given site on the RNA. Protein-RNA cross-linking is a well-established technique for investigating this, but the identification of a cross-linked protein has so far relied upon the availability of antibodies for immunoprecipitation or Western blot studies. To facilitate identification of proteins independent of these techniques, site-specific protein-RNA cross-links were purified in a large scale, which were then used for mass spectrometry (MS). This approach was carried out by the use of a minimal pre-mRNA construct containing a single photoactivatable azidophenacyl group and an adjacent biotin-dT tag for affinity purification of the cross-linked product. To test the feasibility of the method, we purified cross-links to nucleotide 9 downstream of the 5' splice site of pre-mRNA in the spliceosomal complexes A ("pre-spliceosome") and H. By this method, we were able to identify several proteins by MS; the hnRNP proteins A2/B1 were cross-linked to the pre-mRNA in complex A, and FUSE 2/FBP (a homolog of the intronic splicing enhancer KSRP) was cross-linked in complex H.