Conclusion
UV cross-linking lies at the core of several techniques to study RNA interactions. Subsequent formation of covalent bonds ensures maintenance of the complex and facilitates isolation of RNPs. Purifying the transcriptome to capture protein-RNA interactions has spurred the characterisation of the RBPome in an increasing number of microorganisms. Inevitably, the chance of recovering enriched adducts between proteins and hypothetical transcript targets are higher when whole-cell RNA species are used as partitioning baits. Since transcriptome-wide indexing of RBPs is more likely to yield false positives, it is crucial to validate putative RNA-binding activity using protein-centric approaches.
The advantage of producing validatory RNA maps is two-fold. Firstly, interpreting RBP binding profiles can help to unravel their role and its underlying mechanism. Secondly, considering that harsher purification is generally possible in UV requiring methods, these tend to produce less background than their non-UV dependent counterparts. However, cross-linking efficiency and biases may result in false positive signals arising from phosphorylation of DNA or the RBP itself. Furthermore, it is also plausible that the abundance, localisation, or function of a given protein foster consistent but inconsequential RNA cross-linking. As discussed, these situations should become apparent in appropriate downstream functional explorations. Nevertheless, including the mentioned control experiments could economise the efforts invested in characterising proteins which were circumstantially forming duplexes with RNA in previous datasets.
Methodological expansion of these protocols could provide insight into the biology of other components of the RNA interactome. As an illustration, we have referred to CLASH and related protocols, which have specified several RNA-RNA interactions as well as their regulatory functions. Despite not having been reviewed here, an evolving array of techniques has also been using cross-linking and immunoprecipitation to study how the distribution of RNA modifications shape RBP occupancy56,57. Ultimately, this interplay of experimental tools will empower a confident understanding of protein-RNA interactions and their implications in wider biological systems.