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Abstract

RNA–protein interactions play a crucial role in gene expression. These interactions take place in so-called ribonucleoprotein (RNP) complexes. To investigate which proteins interact with RNA in these complexes, and how they do so, UV-light-induced cross-linking has proven to be a valuable, yet straightforward technique. UV irradiation induces a covalent bond between the RNA and the proteins, whereafter cross-linked proteins can be identified by mass spectrometric (MS) approaches. Moreover, the cross-linked region of the protein, and often the actual cross-linked amino acid, can be identified by state-of-the-art MS, as can the cross-linked RNA moiety. This protocol describes in detail how to isolate peptide–RNA oligonucleotide cross-links from UV-irradiated human pre-mRNA RNPs and to perform the subsequent MS investigation of these peptide–RNA conjugates in combination with a dedicated computational analysis, in order to obtain sequence information about the cross-linked peptide and oligoribonucleotide. The described workflow can be applied to any RNP, irrespective of its origin, e.g., RNPs assembled in vitro (as described here) or RNPs isolated from UV-irradiated cells, either ex vivo or in vivo.