In summary, while G2 and G6 in wild type FOXRBE cross-linked to FOXRRM, guanosine did not cross-link efficiently at any other of the other five locations in the FOXRBE. Similarly, uridine readily cross-linked to FOXRRM from position N1 -where A, C and G were unreactive -but not from the four other locations in the FOXRBE. Taken together, the data from this controlled model study confirmed that RNA-protein cross-linking events have strict requirements, beyond simply the proximity of a reactive nucleotide and a reactive amino acid. ). d) SPR traces show that U or G mutations at N3, N4, N5 and N7 of FOXRBE attenuate but do not abolish, FOXRRM binding to FOXRBE variants. The curve for UGCAUGG was fitted using a 1:1 Langmuir binding model including mass-transport limitations. The Kd of the remaining sequences were analysed by a steady state affinity fit curve. e) SDS-PAGE gels show that FOXRBE variants cross-link to FOXRRM with increasing radiation dose. f) CLIR-MS analysis of singly-labelled FOXRBE variants show that protein-RNA cross-linking does not occur with U or G nucleotides located at N3, N4, N5 and N7 (see Table ). The clean-up, enrichment and LC-MS/MS analysis was performed according to the CLIR-MS protocol. The xQuest search was carried out for the masses of mono-to tetranucleotides with a defined mass shift of 5 Da. The data was filtered for mononucleotides (*N indicates a 13 C-labelled nucleotide).