A qualitative and quantitative framework for the characterization of protein-RNA interactions by cross-linking mass spectrometry (XL-MS)

Protein-RNA interactions regulate nearly all levels of gene expression. Their characterization at the molecular basis is critical for understanding the function of protein-RNA complexes, both in cell homeostasis and pathology. Yet, molecular interactions underlying the formation of these complexes remain understudied. UV cross-linking mass spectrometry (XL-MS) is one of the leading approaches for in vitro identification of protein-RNA contact sites, and for providing structural information in the form of distance constraints crucial for the refinement of structural models. Our previously published CLIR-MS method enables qualitative characterization of protein-RNA interactions at residue-specific resolution but was not designed for highly accurate quantitative interpretation. Here, we present a XL-MS workflow based on irradiation with UV light that combines data-dependent acquisition (DDA) and parallel reaction monitoring (PRM) for simultaneous detection and characterization of protein-RNA interaction sites at residue-specific resolution, followed by label-free quantification of cross-linked products. We successfully apply our combined workflow to in vitro reconstituted protein-RNA complexes formed by the proteins TDP-43 and FOX1. We report on novel RNA interaction sites located in the oligomerization interface of the N-terminal domain (NTD) of TDP-43, and characterize quantitatively the process of RNA-induced monomerization of the NTD in an RNA titration experiment. We also investigate temperature-dependent differences in the efficiency of cross-link formation in the FOX1 complex. Our framework offers a unique and novel approach to comparative quantification of protein-RNA cross-links, overcoming the limitations imposed by conventional UV XL-MS methods.