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.
Highlights
Posttranscriptional regulation is an essential part of gene expression control (Buccitelli and Selbach, 2020), and RNA-binding proteins (RBPs) are important players (Gehring et al, 2017; Gebauer et al, 2020)
We focus on UV-crosslinking-based RNA-interactome capture (RIC)-like experiments (Figure 1), some points raised are relevant for cross-linking and immunoprecipitation (CLIP)-like assays and other cross-linking approaches
While the number of known RBPs exceeds the number of known transcription factors, we are just beginning to understand how cell signaling and posttranslational modification (PTM) affect their function
Summary
Posttranscriptional regulation is an essential part of gene expression control (Buccitelli and Selbach, 2020), and RNA-binding proteins (RBPs) are important players (Gehring et al, 2017; Gebauer et al, 2020). Instead of increasing the mRNA-bound protein fraction as suggested by the higher abundance in RIC-like experimental results, mTORC1induced phosphorylation might instead shift LARP1 binding preference to mRNA targets with different isolation and/or crosslinking efficiency. It is very difficult to predict fold changes in RIClike experiments following subcellular localization regulation of RBPs by PTMs. Protein–Protein Interaction and Complex Formation An important function of many RBPs is to bring target RNAs in contact with core ribonucleoprotein machineries, like the exosome, the ribosome and the spliceosome (Gehring et al, 2017). Another example is phosphorylation of UPF1, which triggers formation of the RNA-decay complex and degradation of UPF1-bound RNAs (Durand et al, 2016) In both cases, phosphorylation is expected to affect pulldown efficiencies in RIC-like experiments without necessarily changing RNAbinding. Even though RBPs might interact more with RNAs in condensates, it is not clear if this results in corresponding changes in RIC-like experiments
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