Structural requirements for photo-induced RNA-protein cross-linking

25 June 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Understanding structure-function relationships of RNA-binding proteins requires knowledge of how they bind RNAs in vivo. RNA-protein interactions are studied using light-induced cross-linking at “zero-distance”, yielding nucleotide/amino-acid adducts for mass-spectrometry (MS)-based characterization. However, prerequisites for cross-linking are poorly understood, limiting interpretation of cross-linking data. Here, we report novel insights on cross-linking requirements from studying RBFOX-RRM domain bound to 13C-labeled variants of its heptaribonucleotide binding element as a model. We probed the influence of nucleotide identity, sequence position and amino-acid composition using tandem-MS to assign cross-links at site-specific resolution. We observed cross-linking at three nucleotides, which were stacked onto phenylalanines. Surprisingly, this stacking was required for neighbouring amino-acids to cross-link, and is apparent in published RNA-protein datasets. We hypothesize that π-stacking activates cross-linking via electron transfer, whereafter nucleotide- and peptide radicals, possibly stabilized by capto-dative effects, recombine. These findings should facilitate interpretation of cross-linking data from structural studies and genome-wide datasets.


RNA binding proteins
UV cross linking
mass spectrometry


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