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

Abstract

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.