Investigation of protein-RNA interactions by UV cross-linking and mass spectrometry: methodological improvements toward in vivo applications

Protein–RNA complexes play key roles in a variety of cellular functions such as gene expression and its regulation. One frequently applied method for the investigation of direct protein–RNA interactions is UV induced cross-linking. Mass spectrometry (MS) is increasingly utilized for the identification of proteins and peptides directly interacting with RNA. The major challenges for this application are the generally low yield of the UV induced cross-linking reaction and the lack of suitable tools for MS data analysis. In this work, both issues are adressed. A novel cross-linking pathway involving net loss of H2S from the photoreactive base-analogue 4-thio-uracil was identified, and a novel approach for automated identification of cross-linked peptides from mass spectrometry data was developed