We employed a systematic approach in an effort to identify key structural requirements for RNAprotein cross-linking. We first used C-labelled versions of FOXRBE in a CLIR-MS protocol to identify all points of reaction between the RNA and the protein. We then synthesized mutated C-labelled variants of FOXRBE to determine how cross-linking varies with respect to i) the type of the individual nucleobase, ii) their positions in the RBE, and iii) how it is affected by amino acid composition in the FOXRRM binding site. We were mindful of the fact that mutating key sites in the RNA and the protein might alter the mode of (or even abolish) RNA-protein binding, and therefore for each variant we measured the binding affinity to FOXRRM using surface plasmon resonance spectroscopy (SPR). ). (*N indicates a 13 C-labelled nucleotide).c) CLIR-MS results of b) filtered for mononucleotides showing the site-specific reactivity at U1, G2 and G6 (see Table ). d) FOXRRM/FOXRBE structure showing two cross-linking clusters centered on F126 and U1 and G2 (upper panel), and the cluster at F160 and G6 (lower panel) based on the structure of Auweter et al. . Structures were visualized with PyMOL (The PyMOL Molecular Graphics System, Version 2.5 Schrödinger, LLC).