Identification of protein-RNA contact sites after cross-linking
We have a long standing interest in the analysis of protein-RNA interaction sites in UV irradiated ribonucleoprotein (RNP) particles. Over the last years we developed several strategies for the analysis of cross-linking experiments. These include enrichment strategies making use of titanium dioxide, the employing photoreactive base-analogues (4-thio-uridine, 6-thio-guanine) for the enhancement of cross-linking yields as well as a novel data analysis strategy.
Recent cross-linking projects investigated the protein-RNA interaction between the yeast ASH1 mRNA and the proteins She2p and She3p , human U1 snRNA and snurportin 1 , and box C/D sRNA and Nop5 from Pyrococcus furiosus. Furthermore, cross-linking of human U1 and U2 spliceosomal snRNPs was studied extensively (manuscript in preparation). In addition it was shown that 4-thio-uridine can be used to enhance the cross-linking yield, while some of its cross-linking products are different from those of native RNA.
In our data analysis approach, we make use of the fact that CID/HCD spectra of cross-linked heteroconjugates are dominated by peptide fragments. By subtraction of all possible RNA masses from the experimental precursor mass, the cross-linked peptide can be identified by standard database search.