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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.

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