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

Abstract

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.

A computationally-enhanced hiCLIP atlas reveals Staufen1 RNA binding features and links 3’ UTR structure to RNA metabolism

“…3B). As expected, there is a sharp trough in base-pairing probability around position 0, consistent with UV-crosslinking generally being much more efficient at unpaired nucleotides (25). Strikingly, there is an “M”-shaped profile in the +10 to +75 nt region (shaded grey), that reflects a tendency for a paired-unpaired-paired secondary structure motif downstream of the peak start, viz.…”

Chemical RNA Cross-Linking: Mechanisms, Computational Analysis, and Biological Applications

“…Converting RNA to cDNA fragments is straightforward, but is impacted by side reactions of cross-linking, which reduce efficiency and accuracy of detecting the cross-linking events. Although more resistant to UV damage than DNA, UV-induced lesions in RNA are commonly observed and include base oxidation (primarily 8-oxoguanine), pyrimidine dimers, , and adducts with proteins , and other cellular components (Figure ). Nitrogen mustards are highly reactive alkylating agents that can cross-link nucleic acids to proteins (Figure ).…”

Improved analysis of (e)CLIP data with RCRUNCH yields a compendium of RNA-binding protein binding sites and motifs

“…To better understand how these patterns relate to the specific interaction of an RBP with RNAs, we carried out a simulation of an RBP binding to its cognate motif, crosslinking to the RNA and protecting an extended region of the target from digestion. As the efficiency of crosslinking depends on the identity of both the nucleotides and the amino acids that participate in the RNA-RBP interaction [38], we simulated three scenarios, corresponding to the nucleotide with the highest efficiency of crosslinking being located upstream, within or downstream of the RBP-specific binding motif. For each of these cases we simulated scenarios where the probability of reverse transcriptase reading through the crosslinked nucleotide is very low, intermediate or very high (Fig.…”