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.