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

Nat Commun. 2022 May 17;13(1):2719. doi: 10.1038/s41467-022-30284-w.


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 π-stacked to phenylalanines. Notably, this π-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 π-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids* / chemistry
  • Cross-Linking Reagents / chemistry
  • Immunoprecipitation
  • Nucleotides*
  • Proteins
  • RNA / metabolism


  • Amino Acids
  • Cross-Linking Reagents
  • Nucleotides
  • Proteins
  • RNA