Mass-spectrometric analysis of proteins cross-linked to 4-thio-uracil- and 5-bromo-uracil-substituted RNA

Abstract

UV-induced cross-linking of protein–RNA complexes is widely used to study protein–RNA interactions. Mass spectrometry (MS) has been used successfully to characterise the cross-links obtained. The relatively low yield of the cross-linking reaction can be increased by using photoreactive base-analogues such as 4-thio-uracil and halopyrimidines such as 5-bromo-uracil. However, little is known about the reaction mechanism of 4-thio-uracil with amino acid side chains upon UV light exposure. Therefore the exact mass of the cross-linking product could not be predicted. In this study we performed a mass-spectrometric analysis of proteins that were cross-linked to RNA that contained 4-thio-uridine or 5-bromo-uridine. We observed the formation of three different species from 4-thio-uridine upon UV cross-linking, two indicating reaction pathways differing from the purely additive cross-linking of natural nucleotides. One cross-link originating from 5-bromo-uridine was identified. In parallel, we developed a software for the automated analysis of MS data derived from cross-linking experiments that makes use of commercially available search engines. Thus our study paves the way for future cross-linking experiments with 4-thio-uracil in more complex systems.