Opportunities and Challenges in Global Quantification of RNA-Protein Interaction via UV Cross-Linking

Abstract

RNA-binding proteins (RBPs) are key mediators of posttranscriptional gene expression control. However, the links between cell signaling on the one hand and RBP function on the other are understudied. While thousands of posttranslational modification (PTM) sites on RBPs have been identified, their functional roles are only poorly characterized. RNA-interactome capture (RIC) and cross-linking and immunoprecipitation (CLIP) are attractive methods that provide information about RBP-RNA interactions on a genome-wide scale. Both approaches rely on the in situ UV cross-linking of RBPs and RNAs, biochemical enrichment and analysis by RNA-sequencing (CLIP) or mass spectrometry (RIC). In principle, RIC- and CLIP-like methods could be used to globally quantify RBP-RNA interactions in response to perturbations. However, several biases have to be taken into account to avoid misinterpretation of the results obtained. Here, we focus on RIC-like methods and discuss four key aspects relevant for quantitative interpretation: (1) the RNA isolation efficiency, (2) the inefficient and highly variable UV cross-linking, (3) the baseline RNA occupancy of RBPs, and (4) indirect factors affecting RBP-RNA interaction. We highlight these points by presenting selected examples of PTMs that might induce differential quantification in RIC-like experiments without necessarily affecting RNA-binding. We conclude that quantifying RBP-RNA interactions via RIC or CLIP-like methods should not be regarded as an end in itself but rather as starting points for deeper analysis.

Proteome-wide quantitative RNA-interactome capture identifies phosphorylation sites with regulatory potential in RBM20

“…The defining feature of RBPs is their ability to bind RNA. UV crosslinking can "freeze" such interactions in situ for subsequent protein-or RNA-centric analyses (Vieira-Vieira and Selbach, 2021). Protein-centric methods such as cross-linking and immunoprecipitation (CLIP) involve purification of an individual RBP and identification of its RNA binding sites by sequencing (Wheeler et al, 2018;Lin and Miles, 2019).…”

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

“…Reactions are biased towards uridines and guanosines 19,36,37 , but most amino acids can participate 18,19 . However, cross-links typically only occur at specific positions in the RNA-RBP motif, for which there is currently no mechanistic rationale 38 .…”

Proteome-wide quantitative RNA interactome capture (qRIC) identifies phosphorylation sites with regulatory potential in RBM20

“…3) actually causes nuclear retention of the protein and thereby decreases mRNA-binding in the cytosol indirectly (H. H. Kim et al ., 2008; Grammatikakis, Abdelmohsen and Gorospe, 2017). It is also salient to bear in mind that pull-down efficiencies are affected by factors such as UV cross-linking and mRNA isolation with oligo(dT) beads and therefore do not provide a direct read-out for mRBP-mRNA interaction (Vieira-Vieira and Selbach, 2021). Hence, while qRIC efficiently prioritizes modification sites with regulatory potential, establishing causal relationships still requires follow-up experiments.…”