Nucleotide-amino acid π-stacking interactions initiate photo cross-linking in RNA-protein complexes

Photo-induced cross-linking is a mainstay technique to characterize RNA-protein interactions. However, UV-induced cross-linking between RNA and proteins at zero-distance is poorly understood. Here, we investigate cross-linking of the RBFOX alternative splicing factor with its hepta-ribonucleotide binding element as a model system. We examine the influence of nucleobase, nucleotide position and amino acid composition using CLIR-MS technology (crosslinking-of-isotope-labelled-RNA-and-tandem-mass-spectrometry), that locates cross-links on RNA and protein with site-specific resolution. Surprisingly, cross-linking occurs only at nucleotides that are pi-stacked to phenylalanines. Notably, this pi-stacking interaction is also necessary for the amino-acids flanking phenylalanines to partake in UV-cross-linking. We confirmed these observations in several published datasets where cross-linking sites could be mapped to a high resolution structure. We hypothesize that pi-stacking to aromatic amino acids activates cross-linking in RNA-protein complexes, whereafter nucleotide and peptide radicals recombine. These findings will facilitate interpretation of cross-linking data from structural studies and from genome-wide datasets generated using CLIP (cross-linking-and-immunoprecipitation) methods. Although UV-induced cross-linking is a widely used method to study RNA-protein complexes, the cross-linking reactions are poorly understood. Here, the authors show that pi-stacking interactions between nucleobases and aromatic amino acids play a key role in the cross-linking process.

Automated summary

This study reveals the crucial role of pi-stacking interactions between nucleobases and aromatic amino acids in UV-induced cross-linking of RNA-protein complexes. These findings can contribute to the interpretation of cross-linking data in structural studies and genome-wide datasets.